Methods to treat diabetes and related conditions based on polymorphisms in the tcf-1 gene

ABSTRACT

This invention relates to the use of the novel association between the 483 A&gt;G single nucleotide polymorphism of the TCF1 gene and the clinical response to glycemic control agents, such as DPPIV inhibitors, in patients with disorders of glycemic control, especially diabetes and impaired glucose metabolism. This invention provides methods to classify patients for treatment and/or for optimization of clinical studies and to treat patients based on this association.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods to treat disorders characterized byimpaired glycemic control, especially Diabetes Mellitus and relatedconditions. In particular, this invention relates to the use of genomicanalysis to determine a subject's responsiveness to glycemic controlagents such as dipeptidylpeptidase IV (DPP4) inhibitors and otherglycemic control methods and strategies, including the timing ofinitiation of treatment and the selection of optimum agents. treatmentregimens, and dosages.

2. Description of the Related Art

Diabetes Mellitus is one form of a broad group of disorders in humans,characterized by impaired glycemic control or impaired control of bloodglucose levels. Diabetes itself is a chronic hormonal disordercharacterized by impaired metabolism of glucose and other energyyielding fuels, as well as the late development of serious vascular andneuropathic complications. Diabetes accounts for nearly 15% ofhealthcare costs in the U.S. and is the leading cause of blindness amongworking-age people as well as end-stage renal disease (ESRD) andnon-traumatic limb amputations. Diabetes increases the risk of cardiac,cerebral and peripheral vascular disease 2- to 7-fold and it is a majorcause of neonatal morbidity and mortality.

Diabetes is a complex and diverse group of disorders but all forms areassociated with a common hormonal defect, i.e., insulin deficiency. Thisdeficiency may be total, partial or relative when viewed in the contextof co-existing insulin resistance. Relative or absolute insulindeficiency plays a primary role in the metabolic derangement linked todiabetes and the resulting hyperglycemia in turn plays a key role in thenumerous complications of the disease.

Classification

The newly revised classification of diabetes mellitus is summarized inTable 1. Clinical diabetes may be divided into four general subclasses,including (1) type 1 (caused by beta cell destruction and characterizedby absolute insulin deficiency), (2) type 2 (characterized by insulinresistance and relative insulin deficiency), (3) other specific types ofdiabetes (associated with various identifiable clinical conditions orsyndromes), and (4) gestational diabetes mellitus. In addition to theseclinical categories, two conditions—impaired glucose tolerance andimpaired fasting glucose—refer to a metabolic state intermediate betweennormal glucose homeostasis and overt diabetes. These conditionssignificantly increase the later risk of diabetes mellitus and may insome instances be part of its natural history. It should be noted thatpatients with any form of diabetes might require insulin treatment atsome point. For this reason the previously used terms insulin-dependentdiabetes (for type 1 diabetes mellitus) and non-insulin-dependentdiabetes (for type 2) have been eliminated. TABLE 1 Classification ofdiabetes Clinical diabetes I. Type 1 diabetes, formerly calledinsulin-dependent diabetes mellitus (IDDM) or “juvenile-onset diabetesA. Immune mediated B. Idiopathic II. Type 2 diabetes, formerly callednon-insulin-dependent diabetes (NIDDM) or “adult-onset diabetes” III.Other specific types A. Genetic defects of β-cell function (e.g.,maturity-onset diabetes of the young [MODY] types 1-3 and pointmutations in mito- chondrial DNA) B. Genetic defects in insulin actionC. Disease of the exocrine pancreas (e.g., pancreatitis, trauma,pancreatectomy, neoplasia, cystic fibrosis, hemocrhomatosis,fibrocalculous pancreatopathy) D. Endocrinopathies (e.g., acromegaly,Cushing's syndrome, hyper- thyroidism, pheochromocytoma, glucagonoma,somatostinoma, aldosteronoma) E. Drug or chemical induced (e.g.,glucocorticosteroids, thiazides, diazoxide, pentamidine, vacor, thyroidhormone, phenytoin [Dilantin], β-agonists, oral contraceptives) F.Infections (e.g., congenital rubella, cytomegalovirus) G. Uncommon formsof immune-mediated diabetes (e.g., “stiff- man” syndrome, anti-insulinreceptor antibodies) H. Other genetic syndromes (e.g., Down,Klinefelter's, Turner's syndrome, Huntington's disease, myotonicdystrophy, lipo- dystrophy, ataxia-telangiectasia) IV. Gestationaldiabetes mellitus Risk categories I. Impaired fasting glucose II.Impaired glucose toleranceType 1 Diabetes Mellitus

Patients with this disorder have little or no insulin secretory capacityand depend on exogenous insulin to prevent metabolic decompensation(e.g., ketoacidosis) and death. Commonly but not always, diabetesappears abruptly (i.e., over days and weeks) in previously healthynon-obese children or young adults; in older age groups it may have amore gradual onset. At the time of initial evaluation the typicalpatient often appears ill, has marked symptoms (e.g., polyuria,polydipsia, polyphagia, and weight loss), and may demonstrateketoacidosis. Type 1 diabetes is believed to have a long asymptomaticpre-clinical stage often lasting years, during which pancreatic betacells are gradually destroyed by an autoimmune attack that is influencedby HLA and other genetic factors, as well as the environment. Initially,insulin therapy is essential to restore metabolism toward normal.However, a so-called honeymoon period may follow and last weeks ormonths, during which time smaller doses of insulin are required becauseof partial recovery of beta cell function and reversal of insulinresistance caused by acute illness. Thereafter, insulin secretorycapacity is gradually lost (over several years). The association of type1 diabetes with specific immune response (HLA) genes and the presence ofantibodies to islet cells and their constituents provides strong supportfor the theory that type 1 diabetes is an autoimmune disease. Thissyndrome accounts for less than 10% of diabetes in the United States.

Type 2 Diabetes Mellitus

Type 2, by far the most common form of the disease, is found in over 90%of the diabetic patient population. These patients retain a significantlevel of endogenous insulin secretory capacity. However, insulin levelsare low relative to the magnitude of insulin resistance and ambientglucose levels. Type 2 patients are not dependent on insulin forimmediate survival and ketosis rarely develops, except under conditionsof great physical stress. Nevertheless, these patients may requireinsulin therapy to control hyperglycemia. Type 2 diabetes typicallyappears after the age of 40 years, has a high rate of genetic penetranceunrelated to HLA genes, and is associated with obesity. The clinicalfeatures of type 2 diabetes may be mild (fatigue, weakness, dizziness,blurred vision, or other non-specific complaints may dominate thepicture) or may be tolerated for many years before the patient seeksmedical attention. Moreover, if the level of hyperglycemia isinsufficient to produce symptoms, the disease may become evident onlyafter complications develop.

Other Specific Type of Diabetes

This category encompasses a variety of diabetic syndromes attributed toa specific disease, drug, or condition. Genetic research has providednew insights into the pathogenesis of MODY, which was formerly includedas a form of type 2 diabetes. MODY encompasses several genetic defectsof beta cell function, among which mutations at several genetic loci ondifferent chromosomes have been identified. The most common forms—MODYtype 3—is associated with a mutation for a transcription factor encodedon chromosome 12 named hepatocyte nuclear factor 1α (HNF1, also known asTCF1) and—MODY type 2 is associated with mutations of the glucokinasegene (on chromosome 7). Mutations of the HNF-4α gene (on chromosome 20)are responsible for type 1 of MODY. Each of these conditions isinherited in an autosomal dominant pattern. Two new rare forms of MODYare associated with mutations of the HNF-1β (on chromosome 17) and aninsulin gene transcription factor termed PDX-1 or 1DX-1 (on chromosome13).

The distinction between the various subclasses of diabetes mellitus isusually made on clinical grounds. However, a small subgroup of patientsare difficult to classify, that is, they display features common to bothtype 1 and 2 diabetes. Such patients are commonly non-obese and havereduced insulin secretory capacity that is not sufficient to make themketosis prone. Many initially respond to oral agents but, with time,require insulin. Some appear to have a slowly evolving form of type 1diabetes, whereas others defy easy categorization.

Gestational Diabetes

The term gestational diabetes describes women with impaired glucosetolerance that appears or is first detected during pregnancy.Gestational diabetes usually appears in the 2^(nd) or 3^(rd) trimester,a time when pregnancy-associated insulin antagonistic hormones peak.After delivery, glucose tolerance generally (but not always) reverts tonormal.

Diagnosis

The diagnosis of diabetes is usually straightforward when the classicsymptoms of polyuria, polydipsia, and weight loss are present. All thatis required is a random plasma glucose measurement from venous bloodthat is 200 mg/dL or greater. If diabetes is suspected but not confirmedby a random glucose determination, the screening test of choice isovernight fasting plasma glucose level. The diagnosis is established iffasting glucose is equal to or greater than 126 mg/dL on at least twoseparate occasions.

Related Conditions

Impaired Glucose Tolerance and Impaired Fasting Glucose

Impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) areterms applied to individuals who have glucose levels that are higherthan normal, (under fed or fasting conditions, respectively) but lowerthan those accepted as diagnostic for diabetes mellitus. Both conditionsare associated with an increased risk for cardiovascular disease, but donot produce the classic symptoms or the microvascular and neuropathiccomplications associated with diabetes mellitus. In a subgroup ofpatents (about 25 to 30%), however, type 2 diabetes eventually develops.

Impaired Glucose Metabolism

Impaired Glucose Metabolism (IGM) is defined by blood glucose levelsthat are above the normal range but are not high enough to meet thediagnostic criteria for type 2 diabetes mellitus. The incidence of IGMvaries from country to country, but usually occurs 2-3 times morefrequently than overt diabetes. Until recently, individuals with IGMwere felt to be pre-diabetics, but data from several epidemiologicalstudies argue that subjects with IGM are heterogeneous with respect totheir risk of diabetes and their risk of cardiovascular morbidity andmortality. The data suggest that subjects with IGM, in particular, thosewith impaired glucose tolerance (IGT), do not always develop diabetes,but whether they are diabetic or not, they are, nonetheless, at highrisk for cardiovascular morbidity and mortality. Among subjects withIGM, about 58% have Impaired Glucose Tolerance (IGT), another 29% haveimpaired fasting glucose (IFG), and 13% have both abnormalities(IFG/IGT). As discussed above, IGT is characterized by elevatedpost-prandial (post-meal) hyperglycemia while IFG has been defined bythe ADA on the basis of fasting glycemic values.

The categories of (a) normal glucose tolerance (NGT), (b) impairedglucose metabolism (IGM) and (c) overt type 2 diabetes mellitus weredefined by the ADA in 1997 as follows:

-   (a) Normal Glucose Tolerance (NGT)=fasting plasma glucose level <6.1    mmol/L or less than 110 mg/dl and a 2 h post-prandial glucose level    of <7.8 mmol/L or <140 mg/dl.-   (b) Impaired Glucose Metabolism (IGM) is impaired fasting glucose    (IFG) defined as IFG=fasting glucose level of 6.1-7 mmol/L or    140-220 mg/dl and/or impaired glucose tolerance (IGT)=a 2 h    post-prandial glucose level (75 g OGTT) of 7.8-11.1 mmol/L or    140-220 mg/dl.-   (c) Type 2 diabetes=fasting glucose of greater than 7 mmol/L or 126    mg/dl or a 2 h post-prandial glucose level (75 g OGTT) of greater    than 11.1 mmol/L or 200 mg/dl.

These criteria were defined using the WHO recommended conditions foradministration of an oral glucose tolerance test ((75 g OGTT), i.e., theoral administration of a glucose load containing the equivalent of 75 gof anhydrous glucose dissolved in water with a blood sample taken 2hours later to analyze the post-prandial glucose. Other OGTT testconditions have confirmed the associated risks of the IGT and IFGcategories including: 1) using 50 g glucose instead of 75 g, 2) using acasual (non-fasting) glucose sample as the analyte, and 3) analyzing thepost-prandial glucose at 1 hour rather than 2 hours post-glucose load.Under all of these conditions, the glycemic categories defined abovehave been linked to the increased risks described below, but thestandardized OGTT is preferred in order to minimize variations in testresults.

Individuals with IGM, especially those with the subcategory IFG, areknown to have a significantly higher rate of progression to diabetesthan normoglycemic individuals and are known to be high atcardiovascular risk, especially if they develop diabetes. Interestingly,subjects with IGM, more specifically those with the subcategory IFG,have a high incidence of cancer, cardiovascular diseases and mortalityeven if they never develop diabetes. Therefore, IGM and morespecifically, the subgroup IFG, appears to be at high cardiovascularrisk, especially after patients become overtly diabetic. IGT alsoreferred to as postprandial hyperglycemia (PPHG), on the other hand, isassociated with a high risk for cancer, cardiovascular disease andmortality in non-diabetics and diabetics. See Hanefeld M andTemelkova-Kurktschiev T, Diabet. Med 1997; 14 Suppl. 3: S6-S11.

The increased risk associated with IGT is independent of all other knowncardiovascular risk factors including age, sex, hypertension, low HDLand high LDL cholesterol levels See, Lancet 1999; 354: 617-621. Inaddition, epidemiological studies suggest that postprandialhyperglycaemia (PPHG) or hyperinsulinaemia are independent risk factorsfor the development of macro-vascular complications of diabetesmellitus. See, Mooradian A D and Thurman J E, Drugs 1999; 57(1):19-29.PPHG similiar to HbA1c has been corelated with the presence of diabeticcomplications, notably retinkopathy and nephropathy. See Pettitt D J etal. Lancet 1980; 2: 1050-2, Jarrett R J Lancet 1976; 2: 1009-2 andTeuscher A et al. Diabetes Care 1988; 11: 246-51.

One mechanism through which IGM, and more specifically, IGT, has beenlinked to micro- and macro-angiopathic complications in the absence ofthe abnormal FPG characteristic of diabetics, is postprandialhyperglycemia. Isolated postprandial hyperglycemia, even innon-diabetics, has been shown to reduce the natural free-radicaltrapping agents (TRAP) that are present in serum. Decreasing the levelof TRAP has been shown, under experimental conditions, to be associatedwith an increase in free radical formation and increased oxidativestress. These free radicals have been implicated in the pathologicalmicrovascular and macro-vascular changes associated withatherosclerosis, cardiovascular morbidity and mortality, and cancer See,Ceriello, A, Diabetic Medicine 15: 188-193, 1998. The decrease ofnatural antioxidants like TRAP during post-prandial hyperglycemia mayexplain the increased cardiovascular risk in subjects with IGM, andspecifically IGT, that do not develop diabetes.

The fact that IGT is an independent risk factor in non-diabetics as wellas diabetics justifies it as a new indication, separate from diabetes,for prevention and treatment of cardiovascular morbidity and mortalityas well as cancer. Thus, IGM is associated with following potentialdiseases or conditions: 1) progression to overt diabetes mellitus type 2(Code 250.2 of the International Classification of Diseases 9thversion=ICD-9 Code 250.2) [Diabetes Research and Clinical Practice 1998;40: S 1-S2]; 2) increased microvascular complications of diabetesespecially retinopathy and other ophthalmic complications of diabetes(ICD-9 code 250.5), nephropathy (ICD-9 code 250.4), neuropathy (ICD-9code 250.6) [Diabetes Care 2000, 23: 1113-1118], and peripheralangiopathy or gangrene (ICD-9 code 250.7); 3) increased cardiovascularmorbidity (ICD-9 codes 410-414) especially myocardial infarctions (ICD-9code 410), coronary heart disease or atherosclerosis (ICD-9 code 414)and other acute and subacute forms of coronary ischemia (ICD-9 code411); 4) excess cerebrovascular diseases like stroke (ICD-9 codes430-438) [Circulation 1998, 98:2513-2519]); 5) increased cardiovascularmortality (ICD-9 codes 390-459) [Lancet 1999; 354: 617-621], and suddendeath (ICD-9 code 798.1); 6) higher incidences and mortality rates ofmalignant neoplasms (ICD-9 codes 140-208) [Am J Epidemiol. 1990, 131:254-262, Diabetologia 1999; 42: 1050-1054]. Other metabolic disturbancesthat are associated with IGIVI include dyslipidemia (ICD-9 code 272),hyperuricemia (ICD-9 code 790.6) as well as hypertension (ICD-9 codes401-404) and angina pectoris (ICD-9 code 413.9) [Ann Int Med 1998,128:524-533]. Clearly, the broad spectrum of diseases and conditionsthat are linked to IGM, and especially IGT, represents an area oftremendous medical need.

Many of the same diseases and conditions have been associated with bothIGM and diabetes, but only recently has it been possible to identifythat that the non-diabetic population that has IGM, and especially IGT,should be an indication for prevention and treatment. Accordingly, insubjects with IGM and especially IGT and/or IFG, the restoration ofearly phase insulin secretion and/or the reduction of prandialhyperglycemia should help to prevent or delay the progression to overtdiabetes and to prevent or reduce microvascular complications associatedwith diabetes by preventing the development of the overt diabetes. Inaddition, in individuals with IGM and especially those with IGT and/orIFG, the restoration of early phase insulin secretion and/or reductionof post-prandial hyperglycemia should also prevent or reduce theexcessive cardiovascular morbidity and mortality, and prevent cancer orreduce its mortality in individuals.

insulin Secretion and Action

Insulin is initially synthesized in the pancreatic beta cells as a largesingle-chain polypeptide, pro-insulin, and subsequent cleavage ofpro-insulin results in the removal of a connecting strand (C peptide)and appearance of the smaller, double-chain insulin molecule (51 aminoacid residues). The concentration of glucose is the key regulator ofinsulin secretion. For glucose to activate secretion, it must first betransported by a protein (GLUT 2) into the beta cell, phosphorylated bythe enzyme glucokinase, and metabolized. The immediate triggeringprocess is poorly understood but probably involves the activation ofsignal transduction pathways, closure of adenosine triphosphate(ATP)-sensitive potassium channels, and entry of calcium into the betacell. Normally, when blood glucose rises even slightly above the fastinglevel of 75 to 100 mg/dL, beta cells secrete insulin, initially frompre-formed stored insulin and later from the synthesis of new insulin.The route of glucose entry as well as its concentration determines themagnitude of the response. Higher insulin levels are produced whenglucose is given orally than when given intravenously because of thesimultaneous release of gut peptides (e.g., glucagon-like peptide I,gastric inhibitory polypeptide). Other insulin secretagogues includeamino acids and vagal stimulation. Once secreted into portal blood,insulin removes approximately 50% of the insulin and degrades it. Theconsequence of this uptake is that portal vein insulin is always atleast two- to four-fold higher than that in the peripheral circulation.Conversely, when blood glucose levels decline even slightly (e.g., to 70mg/dL), insulin secretion promptly diminishes.

Insulin acts on responsive tissues by first passing through the vascularcompartment and, on reaching its target, binding to its specificreceptor. The insulin receptor is a heterodimer with two α- and β-chainsformed by disulfide bridges. The α-subunit resides on the extracellularsurface and is the site of insulin binding. The β-subunit spans themembrane and can be phosphorylated on serine, threonine, and tyrosineresidues on the cytoplasmic face. The intrinsic protein tyrosine kinaseactivity of the β-subunit is essential for insulin receptor function.Rapid receptor autophosphorylation and tyrosine phosphorylation ofcellular substrates (e.g., insulin receptor substrates 1 and 2) areimportant early steps in insulin action. Thereafter, a series ofphosphorylation and dephosphorylation reactions are triggered thatultimately produced insulin's effects in insulin-sensitive tissues(liver, muscle, and fat). A variety of post-receptor signal transductionpathways are activated by insulin, including Pl3 (phosphatidylinositol3′) kinase, an enzyme that appears to be critical for the translocationof glucose transporters (GLUT 4) to the cell surface and, in turn,glucose uptake.

A number of other hormones termed counter-regulatory hormones (glucagon,growth hormone, catecholamines, and cortisol) oppose the metabolicactions of insulin. Among these, glucagon and to a lesser extent growthhormone have important roles in development of the diabetic syndrome.Glucagon is secreted by pancreatic alpha cells in response tohypoglycemia, amino acids, and activation of the autonomic nervoussystem. Its major effect is on the liver, where it stimulatesglycogenolysis, gluconeogenesis, and ketogenesis via cyclic adenosinemonophosphate-dependent mechanisms. It is normally inhibited byhyperglycemia but is absolutely or relatively increased in both type 1and type 2 diabetes despite the presence of hyperglycemia.

Diabetes is characterized by marked post-prandial hyperglycemia aftercarbohydrate ingestion. In type 2 diabetes, the combined effects ofdelayed insulin secretion and hepatic insulin resistance impairs thesuppression of hepatic glucose production and the ability of the liverto store glucose as glycogen. Hyperglycemia ensues, even though insulinlevels may eventually rise to levels above those seen in non-diabeticindividuals (insulin secretion remains deficient relative to theprevailing glucose level), because insulin resistance reduces thecapacity of muscle to remove the excess glucose released from the liverand store it in the myocyte as glycogen.

The pharmacological treatment of diabetes mellitus has traditionallyinvolved intervention with insulin or oral glucose-lowering drugs. Intype 1 diabetes, the primary focus is to replace insulin secretion. Intype 2 diabetes, the most well established treatment strategies aim toincrease the secretion or physiological effects of insulin. This can beaccomplished by stimulating insulin secretion directly with insulinsecretogogues such as the sulfonylureas or benzoic acid derivatives, orby reducing peripheral insulin resistance with agents such as thoserepresented by the PPARγ agonist thiazolidinedione class of drugs. Insome type 2 diabetics, insulin itself is needed either early in thestabilization process or in combination with one or more of the otherclasses of drugs. For general review of diabetes see, Cecil Textbook ofMedicine 21^(st) edition; Goldman, L. and Bennett J. C. Eds. SaundersCo. Phili (2000), esp. pages 1263-1285.

Several novel approaches to the treatment of diabetes employ the actionsof Glucagon-Like-Peptide 1 (GLP-1). GLP-1 is a peptide hormone that isreleased into the bloodstream from the intestinal tract in response to ameal. GLP-1 has several actions that lower glucose levels, includingacting directly on pancreatic beta cells to augment insulin release andpromoting the synthesis of insulin. GLP-1 arises from tissue-specificpost-translational processing of the glucagon precursor in theintestinal L-cell, see, Ørskov C. Diabetologia 35:701-711 (1992).

In healthy subjects, GLP-1 potently influences glycemic levels through anumber of physiologic mechanisms including modulation of insulin andglucagon concentrations, see Ørskov C. Diabetologia 35:701-711 (1992);Holst J J, et al. In Glugagon III. Handbook of ExperimentalPharmacology; Lefevbre P J. Ed. Berlin, Springer Verlag, 311-326 (1996);and Deacon C F, et al. Diabetes, Vol. 47:764-769 (1998). The pancreaticeffects of GLP-1 are glucose dependent, see, Kregmann B, et al. Laneifii1300-1304 (1987); Weir G C, Diabetes 38:338-342 (1989).

These same effects also occur in patients with diabetes and tend tonormalize blood glucose levels in type 2 diabetes subjects and improveglycemic control in type 1 patients, see, Gutniak M, et al. N Engl J Med236:1316-1322 (1992); Nathan D M, et al. Diabetes Care 15:270-276(1992); and Nauck M A, et al. Diabetologia 36:741-744 (1993).

Both endogenous and exogenously administered GLP-1 are rapidlymetabolized and have a plasma half-life (t_(1/2)) of only 1-2 minutes invivo. The amino peptidase dipeptidylpeptidase IV (DPP4) is the primarycause of this rapid metabolism. DPP4 action on GLP-1 produces anNH₂-terminally truncated metabolite GLP-1 (9-36) amide, see, Kieffer TJ, et al. Endocrinology 136:3585-3596 (1995); Mentlien R, et al. Eur JBiochem 214:829-635 (1993); Deacon C F, et al. J Clin Endocrinol Metab80:952-957 (1995); Deacon C F, et al. Diabetes 44:1126-1131 (1995).

Dipeptidylpeptidase IV (DPP4; EC 3.4.14.5), is identical to ADAcomplexing protein-2 and to the T-cell activation antigen CD26. DPP4 isa serine exopeptidase that cleaves X-proline dipeptides from theN-terminus of polypeptides. It is an intrinsic membrane glycoproteinanchored into the cell membrane by its N-terminal end. High levels ofthe enzyme are found in the brush-border membranes of the kidneyproximal tubule and of the small intestine, but several other tissuesalso express the enzyme. The enzyme is present in the fetal colon butdisappears at birth. It is ectopically expressed in some human colonadenocarcinomas and human colon cancer cell lines. From such a coloncancer cell line, Darmoul, et al. Ann. Hum. Genet. 54: 191-197, (1990)isolated a cDNA probe for intestinal DPP4 and, by Southern analysis ofsomatic cell hybrids, assigned the gene to chromosome 2. This assignmentwas confirmed by Mathew, et al. Genomics 22: 211-212 (1994), whosublocalized the DPP4 gene to 2q23 by fluorescence in situhybridization. Misumi, et al. Biochim. Biophys. Acta 1131: 333-336,(1992) isolated and sequenced the cDNA coding for DPP4. The nucleotidesequence (3,465 bp) of the cDNA contained an open reading frame encodinga polypeptide comprising 766 amino acids, 1 residue less than those ofthe rat protein. The predicted amino acid sequence exhibited 84.9%identity to that of the rat enzyme.

Abbott, et al. Immunogenetics 40: 331-338 (1994) demonstrated that CD26spans approximately 70 kb and contains 26 exons, ranging in size from 45bp to 1.4 kb. the nucleotides that encode the serine recognition site(G-W-S-Y-G) are split between 2 exons. This clearly distinguishes thegenomic organization of the propyl oligopeptidase family from that ofthe classic serine protease family. CD26 encodes 2 messages sized atabout 4.2 and 2.8 kb. These are both expressed at high levels in theplacenta and kidney and at moderate levels in the lung and liver. Onlythe 4.2 kb mRNA was expressed at low levels in skeletal muscle, heart,brain, and pancreas. By fluorescence in situ hybridization, Abbott, etal. (1994), supra, mapped the gene to 2q24.3.

Any pharmaceutically viable DPP4 (DPP IV) inhibitor can be used toprolong the half-life and increase the action of GLP-1 in vivo. Severalstudies have found that the inhibition of DPP4 improves glucosehomeostasis in rats and augments the in situ response to intravenousglucose load in pigs, see, Deacon F., et al. Diabetes 47:764-769 (1998);Pauly R P, et al. Regal Pept 643:148 (1996); Balkan B, et al.Diabetologia 40(Suppl 1)A131 (1997) and Li X, et al. Diabetes 46(Suppl1):237A (1997).

In pig studies, the inhibition in vivo of DPP4 prevents the NH₂ terminaldegradation of GLP-1, thus extending the t_(1/2) of the biologicallyactive peptide. The presence of the DPP4 inhibitor potentiates both thein-situ response to intravenous glucose given with a GLP-1 infusion andalso improves glucose tolerance seen after oral glucose withoutexogenous GLP-1 by enhancing the action of endogenous GLP-1, see, DeaconC F. Diabetes 47:764-769 (1998).

In other studies targeted inactivation of the DPP4 (or CD26) geneyielded healthy mice that had normal blood glucose levels in the fastedstate but reduced glycemic excursion after a glucose challenge. SeeMarguet D, et al. Proc Natl Acad Sci USA 97:6874-6879 (2000). This groupalso found increased levels of glucose-stimulated circulating insulinand increased intact insulinotropic form of GLP-1 in mice withhomozygous inactivated DPP4 gene.

The administration of a pharmacological inhibitor of DPP4 enzymaticactivity was found to improve glucose tolerance in wild type but not inDPP4 gene inactivated mice. This DPP4 inhibitor was also found toimprove glucose tolerance in mice lacking the gene to produce GLP-1receptors. This suggests that DPP4 inhibition is a valid pharmacologicalapproach that improves blood glucose regulation by controlling theactivity of GLP-1 as well as additional substrates including a relatedincretin hormone, Gastric inhibitory Polypeptide (GIP), see, Marguet D,et al., Supra. Other studies have also shown that pharmacologicalinhibition of DPP4 enzyme activity improves glucose clearance in type 2diabetic animals, see, Deacon C F, et al. Diabetes 47:764 769 (1998);Pederson R A, et al. Diabetes 47:1253-1258 (1998); Paalg R P, et al.Metab-Clin Exp 48:385-389 (1999); and Balkan B. Diabetologia42:1324-1331 (1999). These data reveal the value of DPP4 inhibitors inphysiological glucose homeostasis and the potential for inhibitors orother modulators of DPP4 activity to be effective treatments fordiseases involving altered glucose homeostasis, including diabetes, aswell as conditions capable of being modified by the presence,concentration or activity of the enzyme DPP4.

Agents that inhibit or modify the activity of DPP4 are expected to beunique and useful agents to treat diabetes mellitus and other diseasesin man. At least one DPP4 inhibitor, i.e., 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino ]acetyl ]-, (2S), hasbeen tested in a multicenter, double-blind, randomized, parallelclinical study, comparing the effect of the inhibitor at various doseswith placebo in patients with type 2 diabetes (NIDDM) previously treatedwith diet only, see Ahren B, et al. Diabetes 50(Suppl 2):A104 (2001)

Syndrome-X

Syndrome-X is a metabolic syndrome that is thought to be related todiabetes. The term syndrome-X was given by Reaven et al describing acondition characterized by central obesity, and metabolic manifestationsincluding resistance to insulin stimulated glucose uptake,hyperinsulinemia, glucose intolerance (not necessarily overt diabetes),increased level of very low density lipoprotein triglyceride (VLDL),decreased level of high density lipoprotein cholesterol (HDL)concentrations and hypertension. Each of these characteristic featuresare considered to be risk factors for development of atherosclerosis andother ‘old age’ diseases. It is believed that syndrome-X is caused byinsulin resistance, but no treatment is available at present. See,.Reaven, G. Diabetes. 37:1595-1607, 1988 and Ferrannini, E. et al.Diabetologia. 34:416-422, 1991.

Developments in Molecular Biology and Genetics

During the past two decades, remarkable developments in molecularbiology and genetics have produced a revolutionary growth inunderstanding of the implication of genes in human disease. Genes havebeen shown to be directly causative of certain disease states. Forexample, it has long been known that sickle cell anemia is caused by asingle mutation in the human beta globin gene. In many other cases,genes play a role together with environmental factors and/or other genesto either cause disease or increase susceptibility to disease. Prominentexamples of such conditions include:

-   -   the role of DNA sequence variation in ApoE in Alzheimer's        disease,    -   CKR5 in susceptibility to infection by HIV;    -   Factor V in risk of deep venous thrombosis;    -   MTHFR in cardiovascular disease and neural tube defects;    -   p53 in HPV infection;    -   various cytochrome p450s in drug metabolism;    -   and HLA in autoimmune disease.

Surprisingly, the genetic variations that lead to gene involvement inhuman disease are relatively small. Approximately 1% of the DNA baseswhich comprise the human genome are polymorphic, that is they arevariable between individuals. The genomes of all organisms, includinghumans, undergo spontaneous mutation in the course of their continuingevolution. The majority of such mutations create polymorphisms, thus themutated sequence and the initial sequence co-exist in the speciespopulation. However, the majority of DNA base differences arefunctionally inconsequential in that they neither affect the amino acidsequence of encoded proteins nor the expression levels of the encodedproteins. Some polymorphisms that lie within genes or their promoters dohave a phenotypic effect and it is this small proportion of the genome'svariation that accounts for the genetic component of all differencebetween individuals, e.g., physical appearance, disease susceptibility,disease resistance, and responsiveness to drug treatments. The relationbetween human genetic variability and human phenotype is a central themein modem human genetic studies. The human genome comprises approximately3 billion bases of DNA.

Single Nucleotide Polymorphisms

Sequence variation in the human genome consists primarily of singlenucleotide polymorphisms (“SNPs”) with the remainder of the sequencevariations being short tandem repeats (including micro-satellites), longtandem repeats (mini-satellite) and other insertions and deletions. ASNP is a position at which two alternative bases occur at appreciablefrequency (i.e. >1%) in the human population. A SNP is said to be“allelic” in that due to the existence of the polymorphism, some membersof a species may have the unmutated sequence (i.e., the original“allele”) whereas other members may have a mutated sequence (i.e., thevariant or mutant allele). In the simplest case, only one mutatedsequence may exist, and the polymorphism is said to be diallelic. Theoccurrence of alternative mutations can give rise to triallelicpolymorphisms, etc. SNPs are widespread throughout the genome and SNPsthat after the function of a gene may be direct contributors tophenotypic variation. Due to their prevalence and widespread nature,SNPs have potential to be important tools for locating genes that areinvolved in human disease conditions, see e.g., Wang et al., Science280: 1077-1082 (1998), which discloses a pilot study in which 2,227 SNPswere mapped over a 2.3 megabase region of DNA.

An association between a single nucleotide polymorphisms and aparticular phenotype does not indicate or require that the SNP iscausative of the phenotype. Instead, such an association may indicateonly that the SNP is located near the site on the genome where thedetermining factors for the phenotype exist and therefore is more likelyto be found in association with these determining factors and thus withthe phenotype of interest. Thus, a SNP may be in linkage disequilibrium(LD) with the ‘true’ functional variant. LD, also known as allelicassociation exists when alleles at two distinct locations of the genomeare more highly associated than expected.

Thus a SNP may serve as a marker that has value by virtue of itsproximity to a mutation that causes a particular phenotype.

SNPs that are associated with disease may also have a direct effect onthe function of the gene in which they are located. A sequence variantmay result in an amino acid change or may alter exon-intron splicing,thereby directly modifying the relevant protein, or it may exist in aregulatory region, altering the cycle of expression or the stability ofthe mRNA, see Nowotny P Current Opinions in Neuobiology, 2001,11:637-641.

The role that a common genomic variant might play in susceptibility todisease is best exemplified by the role that the apolipoprotein E (APOE)ε4 allele plays in Alzheimer's disease (AD). The ε4 allele is highlyassociated with the presence of AD and with earlier age of onset ofdisease. It is a robust association seen in many populations studied,see St George-Hyslop et al. Biol Psychiatry 2000, 47:183-199.Polymorphic variation has also been implicated in stroke andcardiovascular disease, see Wu et al. Am J Cardiol 2001, 87; 1361-1366and in multiple sclerosis, see Oksenberg et al. J Neuroimmuol 2001,113:171-184.

It is increasingly clear that the risk of developing many commondisorders and the metabolism of medications used to treat theseconditions are substantially influenced by underlying genomicvariations, although the effects of any one variant might be small.

Therefore, an association between a SNP and a clinical phenotypesuggests, 1) the SNP is functionally responsible for the phenotype or,2) there are other mutations near the location of the SNP on the genomethat cause the phenotype. The 2^(nd) possibility is based on the biologyof inheritance. Large pieces of DNA are inherited and markers in closeproximity to each other may not have been recombined in individuals thatare unrelated for many generations, i.e., the markers are in linkagedisequlibrium (LD).

The available evidence strongly suggests that compounds or therapiesthat modify or inhibit DPP4 activity or otherwise act to improvemetabolic or glycemic control in patients with disorders of impairedglycemic control will be useful in the treatment of disorderscharacterized by impaired glycemic control such as diabetes and otherrelated diseases. These compounds or agents include but are not limitedto the DPP4 inhibitors, 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S) and(1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile).

However, in the past, there has been no way to determine whichindividuals will respond to DPP4 modifiers or other glycemic controlagents and which will not. Thus, there is a need for methods todetermine those individuals who suffer from impaired glycemic control,who will respond to glycemic control agents or therapies, including butnot limited to a DPP4 modifiers or inhibitors or other anti-diabeticagents, or to any agent or therapy intended to improve glycemic control,and those who will not. In addition, there is a need for methods todetermine those individuals, with impaired glycemic control who willrespond to low dose treatment and those individuals who will requirehigher doses to obtain optimal results and therefore custom tailor thetreatment to the individual to provide effective treatment with minimalside effects and danger of drug interaction. In addition, there is aneed for methods to optimize clinical trials of glycemic control agentsor therapies to take into account the significant variation in responsethat these individuals are now known to have.

SUMMARY OF THE INVENTION

The present invention, as described herein below, overcomes deficienciesin currently available methods to treat diabetes with glycemic controlagents or therapies, such as DPP4 modifiers or inhibitors, byidentifying a polymorphism in the TCF1 locus which is associated withthe clinical response to a glycemic control agent or therapy, such as aDPP4 modifier or inhibitor, including but not limited to2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S) and1-[3Hydroxy-adamant-1-ylamino)-acetyl]pyrrolidine-2(S)-carbonitrile. Theidentification of this polymorphism allows the development of a simpletest to determine which patients will respond to DPP4 modifier orinhibitor therapy, including therapy with 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S), or1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile orother GLP-1 based therapies, and therapies acting through othermechanisms of action that tend to normalize glycemic control, and topredict required dosage levels. This will allow the clinician to make amore informed decision about whether or not to treat a patient withdiabetes with a glycemic control agent or therapy such as a DPP4modifier or inhibitor and, if so, how much to use.

These agents and therapies include, but are not limited to, GLP-1 oranalogs thereof including synthetic analogs or natural mimetics,including Exendin-4, and agents activating the GLP-1 receptor, agentsactivating receptors for GIP, PACAP, or glucagon, drugs affectinginsulin secretion or glucose sensing by pancreatic beta cells, includingsulfonylurea agents, meglitinide agents, agents affecting glucokinaseactivity, agents affecting phosphodiesterase activity, agents affectingglucose production or intermediary metabolism including inhibitors ofglucagon secretion or action, modulators of glucocorticoid receptoractivation, biguanides, inhibitors of acetyl CoA carboxylase and otheractivators of fatty acid oxidation, therapies affecting insulin action,including compounds activating or modulating the PPAR family of nuclearhormone receptors, inhibitors of protein phosphatases, inhibitors ofglycogen synthase kinase, inhibitors of the NFkB pathway, SHP2modulators, insulin mimetic agents and biguanides and includingtherapies affecting energy balance, including inhibitors of dietary fatdigestion or absorption (pancreatic lipase, fatty add transport protein,microsomal triglyceride transfer protein, bile acid transporter,diacylglyceride acyltransferase, or pancreatic proteinase inhibitors,and, in addition, therapies affecting carbohydrate digestion, glucoseabsorption or intestinal glucose utilization, including inhibitors ofalpha-glucosidase, inhibitors of amylase and agents delaying gastricemptying such as amylin, or biguanides

Therefore, the present invention provides methods to make use of theTCF-1 genotype of an individual in assessing the utility of glycemiccontrol agents or therapies, including DPP4 inhibitors in the managementof diseases characterized by impaired glycemic control, including: type2 diabetes, type 1 diabetes, impaired glucose tolerance, impairedfasting glucose, Syndrome X, prandial lipemia, hypercholesterolemia,impaired glucose metabolism, gestational diabetes, and abnormal prandialglycemic response (PGR) refering to an excessive or abnormal increase inserum glucose during the prandial period (prandial or post-prandialhyperglycemia).

Thus the present invention provides methods for determining theresponsiveness of an Individual with a disorder characterized byimpaired glycemic control to treatment with a glycemic control agent ortherapy, comprising; determining for the two copies of the TCF1 genepresent in the individual, the identity of the nucleotide pair at thepolymorphic site at 483 A>G, and assigning the individual to a goodresponder group if both pairs are GC or if one pair is AT and one pairis GC and to a low responder group if both pairs are AT.

The method may make use of any glycemic control agents or therapiesincluding, but not limited to, a dipeptidylpeptidase 4 (DPP4) inhibitorsuch as 2-Pyrrolidinecarbonitrile, 1-[[[2-[(5cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S) or1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile orany of the compounds of Formula I or Formula II.

The methods may be used to treat any disorder characterized by impairedglycemic control including, but not limited to; type 2 diabetesmellitus, type 1 diabetes mellitus, impaired glucose tolerance, impairedfasting glucose, Syndrome X, gestational diabetes or any disorderresponsive to DPP4 inhibitors

In another embodiment the present invention provides methods fortreating an individual with a disorder characterized by impairedglycemic control comprising, determining for the two copies of the TCF1gene present in the individual, the identity of the nucleotide pair atthe polymorphic site 483 A>G, wherein, if both the nucleotide pairs areCG or if one is AT and one is CG the individual is treated with aglycemic control agent or therapy and if the nucleotide pairs are bothAT the individual is treated with alternate therapy.

These methods may make use of any glycemic control agents or therapiesincluding but not limited to; a dipeptidylpeptidase 4 (DPP4) inhibitorsuch as 2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S) or1-[(3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile orany of the compounds of Formula I or Formula II.

These methods nay be used to treat any disorder characterized byimpaired glycemic control including, but not limited to, type 2 diabetesmellitus, type 1 diabetes mellitus, impaired glucose tolerance, impairedfasting glucose, Syndrome X, gestational diabetes or any disorderresponsive to DPP4 inhibitors

In a further embodiment the present invention provides methods foridentifying an association between a trait and at least one genotype orhaplotype of the TCF1 gene which comprises, comparing the frequency ofthe genotype or haplotype in a population exhibiting the trait with thefrequency of the genotype or haplotype in a reference population,wherein the genotype or haplotype comprises a nucleotide pair ornucleotide located at the polymorphic site 483 A>G, wherein a higherfrequency of the genotype or haplotype in the trait population than inthe reference population indicates the trait is associated with thegenotype or haplotype. This trait may be, but is not limited to, aclinical response to a drug targeting TCF1 or DPP4.

In a further embodiment the present invention provides methods fortreating an individual, with a disorder characterized by impairedglycemic control, the method comprising, determining for the two copiesof the TCF1 gene present in the individual, the identity of thenucleotide pair at the polymorphic site 483 A>G, wherein, if both thenucleotide pairs are CG or if one is AT and one is CG the individual istreated with a low dose of a glycemic control agent and if thenucleotide pairs are both AT the individual is treated with a high doseof a glycemic control agent.

The above method may make use of any glycemic control agents ortherapies including but not limited to, a dipeptidylpeptidase 4 (DPP4)inhibitor such as 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S) or1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile orany of the compounds of Formula I or Formula II.

The above methods may be used to treat any disorder characterized byimpaired glycemic control including, but not limited to; type 2 diabetesmellitus, type 1 diabetes mellitus, impaired glucose tolerance, impairedfasting glucose, Syndrome X, gestational diabetes or any disorderresponsive to DPP4 inhibitors

In a further embodiment, the present invention provides a method oftreating a patient with a disorder characterized by impaired glycemiccontrol comprising, providing genetic counseling to the patient andpatients family, determining the patients genotype for the TCF1 gene atthe polymorphism site 483 A>G, and then determining the proper therapyfor said patient based on results of the genotype determination.

In a further embodiment the present invention provides a method foroptimizing clinical trial design for glycemic control agents,comprising, determining, for the two copies of the TCF1 gene present inan individual being considered for inclusion in the clinical trial, theidentity of the nucleotide pair at the polymorphic site 483 A>G,wherein, if both the nucleotide pairs are CG or if one is AT and one isCG the individual is included in the clinical trial and if thenucleotide pairs are both AT the individual is not included.

In a further embodiment the present invention provides a method foridentifying individuals, with a disorder characterized by impairedglycemic control, who would benefit from drug A vs. B, comprising,determining, for the two copies of the TCF1 gene present in theindividual, the identity of the nucleotide pair at the polymorphic site483 A>G, wherein, if both the nucleotide pairs are CG or if one is ATand one is CG the individual would benefit from a glycemic control agentor therapy and if the nucleotide pairs are both AT the individual wouldbenefit from an alternate glycemic control agent or therapy.

In a further embodiment the present invention provides a method fordetermining which individuals, with a disorder characterized by impairedglycemic control, could be treated with a glycemic control agents withreduced side effects, comprising, determining, for the two copies of theTCF1 gene present in the individual, the identity of the nucleotide pairat the polymorphic site 483 A>G, wherein, if both the nucleotide pairsare CG or if one is AT and one is CG the individual can be treated withlower doses of a glycemic control agent with fewer side effects and ifthe nucleotide pairs are both AT the individual must be treated withhigher doses of a glycemic control agent and therefore greater sideeffects.

In a further embodiment, the invention provides methods for determiningthe responsiveness of an individual with a disorder characterized byimpaired glycemic control to treatment with a glycemic control agent ortherapy, comprising; determining, for the two copies of the TCF1 genepresent in the individual, the identity of a nucleotide pair at apolymorphic site in the region of the TCF1 gene that is in linkagedisequilibrium with the polymorphic site at TCF1 483 A>G, and assigningthe individual to a good responder group if the nucleotide pair at apolymorphic site in the region of the TCF1 gene that is in linkagedisequilibrium with the polymorphic site at 483 A>G, indicates that, atthe TCF1 polymorphic site at 483 A>G, both nucleotide pairs are GC orone pair is AT and one pair is GC and to a low responder group if saidnucleotide pair indicates that both pairs are AT at the TCF1 483 A>Gsite.

BRIEF DISCUSSION OF THE DRAWING

FIG. 1 is a diagram showing the mean (±SEM) prandial glycemic responsefor each of the alleles of TCF1 for the polymorphism at 483 A>G, i.e.,AG, AA and GG, for subjects treated with placebo or with a DPP-IVinhibitor as described in the text. Levels of significant differencesbetween placebo and inhibitor-treated subjects of the same genotype areindicated within the figure.

FIG. 2 is a diagram showing the mean (±SEM) glycosylated hemoglobin(HbA1c) response for each of the alleles of TCF1 for the polymorphism at483 A>G. i.e., AG, AA and GG for subjects treated with placebo or with aDPP-IV inhibitor as described in the text. Levels of significantdifferences between placebo and inhibitor-treated subjects of the samegenotype are indicated within the figure.

FIG. 3 Shows the sequence of the section of the TCF1 gene where the 483A>G polymorphism is located (SEQ ID NO: 1). This sequence is derivedfrom GenBank accession number U72616. The polymorphic nucleotide islocated at nucleotide No, 183 in SEQ ID NO: 1, and may be A or G. Alsoindicated in this sequence in FIG. 3 are the sequences used for theforward and reverse primers used for PCR amplification. SEQ ID NO: 2 isthe invader probe and Probe 1 and Probe 2 are SEQ ID NOS: 3 and 4respectively. In FIG. 3 the nucleotide marked with * is the nucleotidethat is polymorphic, the nucleotides in bold represent the forward andreverse primers used for PCR amplification and the underlinednucleotides represent the extension primers.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The DPP4 Inhibitor Study

The genotypes of 76 individuals, enrolled in a study of a specificinhibitor of DPP4 in diabetic patients, were examined for polymorphismsin 91 loci in an effort to identify genetic determinants (such as SNPs)or correlates of response to the DPP4 inhibitor being studied, i.e.,2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-(2S). The genetic loci examined included thosegenes thought to be related to the pathway of the anti-diabetic actionof the compound as well as genes thought to be related to the geneticetiology of diabetes. A highly significant relationship (p=0.00051) wasfound between the 48 A>G polymorphism at the TCF1 locus and thetreatment response in the integrated exposure to glucose measured duringa four hour standardized breakfast meal. This response is referred to asthe prandial glycemic response (PGR), see FIG. 1.

The product of the TCF1 gene is TCF1 transcription factor 1, hepatic.This transcription factor is also known as; LF-B1, hepatic nuclearfactor-1 alpha (HNF-1 alpha) and albumin proximal factor and is known toregulate the activation of genes responsible for insulin response.Mutations in the TCF1 gene have been previously associated withsusceptibility to MODY type 3, See, Urhammer S A, Diabetologia 1997,40(4):473-5.

The TCF1 gene is located at chromosome location: 12q24.2. The standardnomenclature for the nucleotide substitution for the polymorphism ofthis invention is 483 A>G and consequent amino acid substitution in theexpressed polypeptide product is Asn 487 Ser. This polymorphism wasreported in 1997, See, Urhammer S A, Diabetologia 1997, 40(4):473-5(PMID: 9112026). The polymorphism is located in the partial sequenceshown in FIG. 3, and was derived from GenBank accession number U72616.

Among the DPP4 treated individuals there was a significant difference inthe prandial glycemic response (PGR) between individuals of the GGgenotype and individuals with the AG or AA genotype with GG homogenouspatients having the best response to 2-Pyrrolidinecarbonitrile,1-[[[2[(5cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S) in thesense of improved glucose homeostasis with treatment.

It is now recognized that prandial glycemic control is one element of anintegrated strategy to reduce complications of diabetes that are thoughtto be driven by the combined increase in glucose exposure during theprandial period as well as from elevated fasting plasma glucoseconcentrations. Any strategy to improve the impact of a given agent onthe overall glycemic control must take into account the need to improvethis integrated exposure.

As used herein, the term “prandial” shall mean during the meal.

As used herein, the term “post-prandial” shall mean during theabsorbtion period following meal intake (approximatly 0-8 hours,depending on the meal sixe and composition).

As used herein, the term “post-absorptive” shall mean after nutrientabsorption is completed or approximatly 4-8 hours post-meal.

As used herein, the term “fasting” shall mean after a prolonged periodi.e. 12-16 hours, without eating.

As used herein, the term “prandial glycemic response” (PGR) refers tothe change in serum glucose during the prandial or post-prandial period.

The level of glycosylated hemoglobin (HbA1c) in circulating erythrocyteshas been firmly established as an integrated marker of glycemic controlthat reflects long-term exposure to glucose concentrations. In thepresent invention, it has been discovered that in addition to therelationship between prandial glycemic response and the GG TCF1genotype, both TCF1 AG and TCF1 GG genotypes are associated with anoverall improvement in glycemic control, evidenced by an association ofthe AG and GG TCF1 genotypes with improved changes in glycosylatedhemoglobin (HbA1c) levels after four weeks of treatment with2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S) (see FIG. 2).

As used herein the term “disorders characterized by impaired glycemiccontrol” (IGC) shall mean a metabolic disorder in which one of theprimary manifestation is the excessive or abnormal elevation of bloodglucose levels, either in the fasting state or in response to a meal oran oral glucose load and shall include; type 2 diabetes, type 1diabetes, impaired glucose metabolism. i.e., impaired glucose tolerance(post-prandial hyperglycemia) and/or impaired fasting glucose, SyndromeX, gestational diabetes and abnormal prandial glycemic response (PGRrefering to an excessive or abnormal increase in serum glucose duringthe prandial period (prandial or post-prandial hyperglycemia).

As used herein, the term “glycemic control agent or therapy” shall meanany compound, drug or form of treatment that, in a patient with; type 2diabetes or type 1 diabetes, impaired glucose tolerance, impairedfasting glucose, Syndrome X, post-prandial hyperglycemia or gestationaldiabetes will tend to normalize fasting, prandial or post-prandial serumglucose levels or to normalize glycosylated hemoglobin (HbA1c) responseover time.

The term “DPP4 inhibitor”, as used herein, means a compound capable ofinhibiting the catalytic actions of the enzyme DPP4 (DPP-IV;dipeptidylpeptidase IV; EC 3.4.14.5), which is a serine exopeptidaseidentical to ADA complexing protein-2 and to the T-cell activationantigen CD26.

Many compounds that act as inhibitors of DPP4 enzyme activity are nowknown, such as 2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S) and(1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile)and including, but not limited to, the compounds disclosed in U.S. Pat.Nos.; 6,011,155, 6,124,305, 6,166,063, 5,602,102, 6,110,949, 6,274,608B1, 5,462,928, 6,172,081, 6,107,317, 6,110,949, 6,172,081, 5,939,560,5,543,396, and 6,107,317 and international Publications WO 01/34594 A1,WO 01/47514 A1, WO 00/34241, WO 01/55085 A1, WO 01/52825 A2, WO 01/04156A1, WO 00/10549, WO 01/55105 A1, WO 99/67278, WO 95/15309, WO 98/19998,WO 01/34594, WO 01/62266, WO 97/40832, WO 01/72290, WO 01/68603, WO00/34241, WO 99/61431, WO 99/67279, WO 93/08259, WO 95/11689, WO91/16339, WO 93/08259, WO 95/11689, WO 95/29691, WO 95/34538, WO99/46272, WO 95/29691, WO 00/53171 and WO 99/38501 and EP1052994,EP1019494, EP0528858, EP0610317, EP1050540, EP1062222 and German PatentsNos. 158109 and 296075, the contents of all of these patents andpublications are hereby incorporated by reference herein for allpurposes. Any of the DPP4 inhibitors disclosed in the above patents andpublications may be used in the methods of the present invention.Particularly preferred DPP4 inhibitors are the compounds2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S) and(1-[3Hydroxy-adamant-1-ylamino)-acetyl-pyrrolidine-2(S)-carbonitrile).

Therefore, the present invention is based, in part, on the discovery ofthe novel association, in patients with disorders characterized byimpaired glycemic control, of genetic variants or single nucleotidepolymorphisms (“SNPs”) of the TCF1 gene with the clinical response toglycemic control agents or therapies including but not limited toadministration of a DPP4 inhibitor.

As described in detail below, these variants are associated withsignificant variation in the clinical response to modifiers orinhibitors of the enzyme DPP4 in the treatment of diabetes and otherdiseases that are responsive to inhibitors or modifiers of the activityof the enzyme DPP4, including therapy with 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S), andother GLP-1 based therapies, and therapies acting through other similarmechanisms of action that tend to stabilize glycemic control. Thesevariants were found in genomic DNAs isolated from 76 individualsparticipating in a study of the effect of the DPP4 inhibitor,2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S), in the treatment of type 2 diabetes(NIDDM).

Formula I Compounds

Other DPP4 inhibitors that may be used in the present invention include,but are not limited to, the following N-(N′-substitutedglycyl)-2-cyanopyrrolidines, these, as a group constitute formula I asdescribed below;

wherein R is:

-   -   a) R₁R_(1a)N(CH₂)_(m)— wherein    -    R₁ is a pyridinyl or pyrimidinyl moiety optionally mono- or        independently disubstituted with (C₁₋₄)alkyl, (C₁₋₄)alkoxy,        halogen, trifluoromethyl, cyano or nitro; or phenyl optionally        mono- or independently disubstituted with (C₁₋₄)alkyl,        (C₁₋₄)alkoxy or halogen; R_(1a) is hydrogen or (C₁₋₈)alkyl; and        m is 2 or 3;    -   b) (C₃₋₁₇)cycloalkyl optionally monosubstituted in the        1-position with (C₁₋₃)hydroxyalkyl;    -   c) R₂(CH₂)_(n)— wherein either    -    R₂ is phenyl optionally mono- or independently di- or        independently trisubstituted with (C₁₋₄)alkyl, (C₁₋₄)alkoxy,        halogen or phenylthio optionally monosubstituted in the phenyl        ring with hydroxymethyl; or is (C₁₋₈)alkyl; a [3.1.1]bicyclic        carbocyclic moiety optionally mono- or plurisubstituted with        (C₁₋₈)alkyl; a pyridinyl or naphthyl moiety optionally mono- or        independently disubstituted with (C₁₋₄)alkyl, (C₁₋₄)alkoxy or        halogen; cyclohexene; or adamantyl; and    -    n is 1 to 3; or    -    R₂ is phenoxy optionally mono- or independently disubstituted        with (C₁₋₄)alkyl, (C₁₋₄)alkoxy or halogen; and    -    n is 1 or 3;    -   d) (R₃)₂CH(CH₂)₂— wherein each R₃ independently is phenyl        optionally mono- or independently disubstituted with        (C₁₋₄)alkyl, (C₁₋₄)alkoxy or halogen;    -   e) R₄(CH₂)_(p)— wherein R₄ is 2-oxopyrrolidinyl or (C₂₋₄)alkoxy        and p is 2 to 4;    -   f) isopropyl optionally monosubstituted in 1-position with        (C₁₋₃)hydroxyalkyl;    -   g) R₅ wherein R₅ is: indanyl; a pyrrolidinyl or piperidinyl        moiety optionally substituted with benzyl; a [2.2.1]- or        [3.1.1]bicyclic carbocyclic moiety optionally mono- or        plurisubstituted with (C₁₋₈)alkyl; adamantyl; or (C₁₋₈)alkyl        optionally mono- or independently plurisubstituted with hydroxy,        hydroxymethyl or phenyl optionally mono- or independently        disubstituted with (C₁₋₄)alkyl, (C₁₋₄)alkoxy or halogen;        in free form or in acid addition salt form.

The compounds of formula I can exist in free form or in acid additionsalt form. Salt forms may be recovered from the free form in knownmanner and vice-versa. Acid addition salts may, e.g., be those ofpharmaceutically acceptable organic or inorganic acids. Although thepreferred acid addition salts are the hydrochlorides, salts ofmethanesulfonic, sulfuric, phosphoric, citric, lactic and acetic add mayalso be utilized.

The compounds of formula 1 may exist in the form of optically activeisomers or diastereoisomers and can be separated and recovered byconventional techniques, such as chromatography.

“Alkyl” and “alkoxy” are either straight or branched chain, of whichexamples of the latter are isopropyl and tert-butyl.

R preferably is a), b) or e) as defined above. R₁ preferably is apyridinyl or pyrimidinyl moiety optionally substituted as defined above.R₁a preferably is hydrogen. R_(1a) preferably is phenyl optionallysubstituted as defined above. R₃ preferably is unsubstituted phenyl. R₄preferably is alkoxy as defined above. R₅ preferably is optionallysubstituted alkyl as defined above. m preferably is 2. n preferably is 1or 2, especially 2. p preferably is 2 or 3, especially 3.

Pyridinyl preferably is pyridin-2-yl; it preferably is unsubstituted ormonosubstituted, preferably in 5-position. Pyrimidinyl preferably ispyrimidin-2-yl. It preferably is unsubstituted or monosubstituted,preferably in 4-position. Preferred as substitutents for pyridinyl andpyrimidinyl are halogen, cyano and nitro, especially chlorine.

When it is substituted, phenyl preferably is monosubstituted; itpreferably is substituted with halogen, preferably chlorine, or methoxy.It preferably is substituted in 2-, 4- and/or 5-position, especially in4-position. (C₃₋₁₂) cycloalkyl preferably is cyclopentyl or cyclohexyl.When it is substituted, it preferably is substituted with hydroxymethyl.(C₁₋₄) alkoxy preferably is of 1 or 2 carbon atoms, it especially ismethoxy. (C₂₋₄) alkoxy preferably is of 3 carbon atoms, it especially isisopropoxy. Halogen is fluorine, chlorine, bromine or iodine, preferablyfluorine, chlorine or bromine, especially chlorine. (C₁₋₄) alkylpreferably is of 1 to 6, preferably 1 to 4 or 3 to 5, especially of 2 or3 carbon atoms, or methyl. (C₁₋₄) alkyl preferably is methyl or ethyl,especially methyl. (C₁₋₄) hydroxyalkyl preferably is hydroxymethyl.

A [3.1.1]bicyclic carbocyclic moiety optionally substituted as definedabove preferably is bicyclo[3.1.1]hept-2-yl optionally disubstituted in6-position with methyl, or bicyclo[3.1.1]hept-3-yl optionallytrisubstituted with one methyl in 2-position and two methyl groups in6-position. A [2.2.1]bicyclic carbocyclic moiety optionally substitutedas defined above preferably is bicyclo[2.2.1]hept-2-yl.

Naphthyl preferably is 1-naphthyl. Cyclohexene preferably iscyclohex-1-en-1-yl. Adamantyl preferably is 1- or 2-adamantyl.

A pyrrolidinyl or piperidinyl moiety optionally substituted as definedabove preferably is pyrrolidin-3-yl or piperidin-4yl. When it issubstituted it preferably is N-substituted.

A preferred group of compounds of formula 1 are the compounds wherein Ris R′ (compounds Ia), whereby R′ is: R₁′NH(CH₂)₂— wherein R₁′ ispyridinyl optionally mono- or independently disubstituted with halogen,trifluoromethyl, cyano or nitro; or unsubstituted pyrimidinyl;(C₃₋₇)cycloalkyl optionally monosubstituted in 1-position with(C₁₋₃)hydroxyalkyl; R₄′(CH₂)₃— wherein R₄′ is (C₂₋₄)alkoxy; or R₅,wherein R₅ is as defined above; in free form or in acid addition saltform.

More preferred compounds of formula I are those wherein R is R′(compounds Ib), whereby R″ is: R₁″NH(CH₂)₂— wherein R₁″ is pyridinylmono- or independently disubstituted with halogen, trifluoromethyl,cyano or nitro; (C₄₋₈)cycloalkyl monosubstituted in 1-position with(C₁₋₃)hydroxyalkyl; R₄′(CH₂)₃— wherein R₄′ is as defined above; or R₅′wherein R₅′ is a [2.2.1]- or [3.1.1]bicyclic carbocyclic moietyoptionally mono- or plurisubstituted with (C₁₋₈)alkyl; or adamantyl; infree form or in acid addition salt form.

Even more preferred compounds of formula I are those wherein R is R′″(compounds Ic), whereby R′″ is: R₁″NH(CH₂)₂— wherein R₁″ is as definedabove; (C₁₋₈)cycloalkyl monosubstituted in 1-position withhydroxymethyl; R₄′(CH₂)₃— wherein R₄′ is as defined above; or R₅″wherein R₅″ is adamantyl; in free form or in acid addition salt form.

A further group of compounds are Ip, wherein R is R^(p), which is:

-   -   a) R₁ ^(p)NH(CH₂)₂— wherein R₁ ^(p)is a pyridinyl or pyrimidinyl        moiety optionally mono- or independently disubstituted with        halogen, trifluoromethyl, cyano or nitro;    -   b) (C₃₋₇)cycloalkyl optionally monosubstituted in 1-position        with (C₁₋₄)hydroxyalkyl;    -   c) R₁ ^(p)(CH₂)₂— wherein R₂ ^(p) is phenyl optionally mono- or        independently di- or independently trisubstituted with halogen        or (C₁₋₃)alkoxy;    -   d) (R₃ ^(p))₂CH(CH₂)₂— wherein each R₃ ^(p) independently is        phenyl optionally monosubstituted with halogen or (C₁₋₃)alkoxy;    -   e) R₄(CH₂)₃— wherein R₄ is as defined above; or    -   f) isopropyl optionally monosubstituted in 1-position with        (C₁₋₃)hydroxyalkyl; in free form or in pharmaceutically        acceptable acid addition salt form.

A further group of compounds are those wherein R is R^(s), which is:

-   -   a) R₁ ^(s)R_(1a) ^(s)(CH₂)_(ms)— wherein R₁ ^(s) is pyridinyl        optionally mono- or independently disubstituted with chlorine,        trifluoromethyl, cyano or nitro; pyrimidinyl optionally        monosubstituted with chlorine or trifluoromethyl; or phenyl;        R_(1a) ^(s) is hydrogen or methyl; and ms is 2 or 3;    -   b) (C₃₋₁₂)cycloalkyl optionally monosubstituted in 1-position        with hydroxymethyl;    -   c) R₂ ^(s)(CH₂)_(ms)— wherein either R₂ ^(s) is phenyl        optionally mono- or independently di- or independently        trisubstituted with halogen, alkoxy of 1 or 2 carbon atoms or        phenylthio monosubstituted in the phenyl ring with        hydroxymethyl; (C₁₋₆)alkyl; 6,6-dimethylbicyclo[3.1.1]hept-2-yl;        pyridinyl; naphthyl; cyclohexene; or adamantyl; and ns is 1 to        3; or R₂ ^(s) is phenoxy; and ns is 2;    -   d) (3,3-diphenyl)propyl;    -   e) R₄ ^(s)(CH₂)_(ps) wherein R₄ ^(s) is 2-oxopyrrolidin-1-yl or        isopropoxy and ps is 2 or 3;    -   f) isopropyl optionally monosubstituted in 1-position with        hydroxymethyl;    -   g) R₅ ^(s) wherein R₅ ^(s) is: indanyl; a pyrrolidinyl or        piperidinyl moiety optionally N-substituted with benzyl;        bicyclo[2.2.1]hept-2-yl; 2,6,6trimethylbicyclo-[3.1.1]hept-3-yl;        adamantyl; or (C₁₋₈)alkyl optionally mono- or independently        disubstituted with hydroxy, hydroxymethyl or phenyl;        in free form or in acid addition salt form.        Formula II Compounds

In addition, other DPP4 inhibitors may be used in the present inventionincluding, but not limited to, the following N-(substituted glycyl)-2-cyanopyrrolidines, these compounds, as a group constitute formula II asdescribed below;

wherein R is substituted adamantyl; and n is 0 to 3; in free form or inacid addition salt form. The compounds of formula II can exist in freeform or in acid addition salt form. Pharmaceutically acceptable (i.e.,non-toxic, physiologically acceptable) salts are preferred, althoughother salts are also useful, e.g., in isolating or purifying thecompounds of this invention. Although the preferred acid addition saltsare the hydrochlorides, salts of methanesulfonic, sulfuric, phosphoric,citric, lactic and acetic acid may also be utilized.

The compounds of the invention may exist in the form of optically activeisomers or diastereoisomers and can be separated and recovered byconventional techniques, such as chromatography.

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group. The term“alkyl” refers to straight or branched chain hydrocarbon groups having 1to 10 carbon atoms, preferably 1 to 7 carbon atoms, most preferably 1 to5 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl,isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl and the like. Theterm “alkanoyl” refers to alkyl-C(O)—. The term “substituted adamantyl”refers to adamantyl, i.e., 1- or 2-adamantyl, substituted by one ormore, for example two, substitutents selected from alkyl, —OR.sub.1 or—NR.sub.2 R.sub.3; where R.sub.1, R. sub.2 and R.sub.3 are independentlyhydrogen, alkyl, (C.sub.1-C.sub.8-alkanoyl), carbamyl, or —CO—NR.sub.4R.sub.5; where R.sub.4 and R.sub. 5 are independently alkyl,unsubstituted or substituted aryl and where one of R.sub.4 and R.sub.5additionally is hydrogen or R.sub.4 and R.sub. 5 together representC.sub.2-C.sub.7 alkylene. The term “aryl” preferably represents phenyl.Substituted phenyl preferably is phenyl substituted by one or more,e.g., two, substitutents selected from, e.g., alkyl, alkoxy, halogen andtrifluoromethyl. The term “alkoxy” refers to alkyl-O—. The term“halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.The term “alkylene” refers to a straight chain bridge of 2 to 7 carbonatoms, preferably of 3 to 6 carbon atoms, most preferably 5 carbonatoms.

A preferred group of compounds of the invention is the compounds offormula I wherein the substituent on the adamantyl is bonded on abridgehead or a methylene adjacent to a bridgehead. Compounds of formulaII wherein the glycyl-2-cyanopyrrolidine moiety is bonded to abridgehead, the R′ substituent on the adamantyl is preferably 3-hydroxy.Compounds of formula II wherein the the glycyl-2-cyanopyrrolidine moietyis bonded at a methylene adjacent to a bridgehead, the R′ substituent onthe adamantyl is preferably 5-hydroxy.

Particularly preferred DPP4 inhibitors are the compounds;2-Pyrrolidinecarbonitrile, 1-[[[2[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S) and1-(3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile.

Thus, in a first aspect, the invention provides methods of determiningthe responsiveness of an individual with; type 2 diabetes, impairedglucose tolerance, impaired fasting glucose, Syndrome X, prandiallipemia, hypercholesterolemia, hypertension, gestational diabetes ortype 1 diabetes or any DPP4 inhibitor responsive disorder, to treatmentwith a DPP4 inhibitor compound or to glycemic control agents ortherapies. These methods comprise determining the genotype or haplotypeof the TCF1 gene and making the determination of responsiveness based onthe presence or absence of one or more polymorphisms in the TCF1 gene.This aspect of the invention also provides methods of determining theresponsiveness of an individual with diabetes or a related metabolicdisorder, to treatment with other agents or therapies intended toimprove metabolic control. The detection of these polymorphisms can beused to determine or predict the responsiveness of the individual to aparticular drug or other therapy. One of skill in the art will readilyrecognize that, in addition to the specific polymorphisms disclosedherein, any polymorphism that is in linkage disequilibrium with the saidpolymorphism can also serve as a surrogate marker indicatingresponsiveness to the same drug or therapy as does the SNP that it is inlinkage disequilibrium with. Therefore, any SNP in linkagedisequilibrium with the SNPs disclosed in this specification, can beused and is intended to be included in the methods of this invention.

Identification and Characterization of SNPs

Many different techniques can be used to identify and characterize SNPs,including single-strand conformation polymorphism analysis, heteroduplexanalysis by denaturing high-performance liquid chromatography (DHPLC),direct DNA sequencing and computational methods, see Shi M M, Clin Chem2001, 47:164-172. Thanks to the wealth of sequence information in publicdatabases, computational tools can be used to identify SNPs in silico byaligning independently submitted sequences for a given gene (either cDNAor genomic sequences). Comparison of SNPs obtained experimentally and byin silico methods showed that 55% of candidate SNPs found bySNPFinder(http://lpgws.nci.nih.gov:82/perl/snp/snp_cgi.pl) have alsobeen discovered experimentally, see, Cox et al. Hum Mutal 2001,17:141-150. However, these in silico methods could only find 27% of trueSNPs.

The most common SNP typing methods currently include hybridization,primer extension and cleavage methods. Each of these methods must beconnected to an appropriate detection system. Detection technologiesinclude fluorescent polarization, (see Chan X et al. Genome Res 1999,9:492-499), luminometric detection of pyrophosphate release(pyrosequencing), (see Ahmadiian A et al. Anal Biochem 2000,280:103-10), fluorescence resonance energy transfer (FRET)-basedcleavage assays, DHPLC, and mass spectrometry, (see Shi M M, Clin Chem2001, 47:164-172 and U.S. Pat. No. 6,300,076 B1). Other methods ofdetecting and characterising SNPs are those disclosed in U.S. Pat. Nos.6,297,018 B1 and 6,300,063 B1. The disclosures of the above referencesare incorporated herein by reference in their entirety.

In a particularly preferred embodiment the detection of the polymorphismcan be accomplished by means of so called INVADER™ technology (availablefrom Third Wave Technologies Inc. Madison, Wis.). In this assay, aspecific upstream “invader” oligonucleotide and a partially overlappingdownstream probe together form a specific structure when bound tocomplementary DNA template. This structure is recognized and cut at aspecific site by the Cleavase enzyme, and this results in the release ofthe 5′ flap of the probe oligonucleotide. This fragment then serves asthe “invader” oligonucleotide with respect to synthetic secondarytargets and secondary fluorescently labeled signal probes contained inthe reaction mixture. This results in specific cleavage of the secondarysignal probes by the Cleavase enzyme. Fluoresence signal is generatedwhen this secondary probe, labeled with dye molecules capable offluorescence resonance energy transfer, is cleaved. Cleavases havestringent requirements relative to the structure formed by theoverlapping DNA sequences or flaps and can, therefore, be used tospecifically detect single base pair mismatches immediately upstream ofthe cleavage site on the downstream DNA strand. See Ryan D et al.Molecular Diagnosis Vol. 4 No 2 1999:135-144 and Lyamichev V et al.Nature Biotechnology Vol 17 1999:292-296, see also U.S. Pat. Nos.5,846,717 and 6,001,567 (the disclosures of which are incorporatedherein by reference in their entirety).

In some embodiments, a composition contains two or more differentlylabeled genotyping oligonucleotides for simultaneously probing theidentity of nucleotides at two or more polymorphic sites. It is alsocontemplated that primer compositions may contain two or more sets ofallele-specific primer pairs to allow simultaneous targeting andamplification of two or more regions containing a polymorphic site.

TCF1 genotyping oligonucleotides of the invention may also beimmobilized on or synthesized on a solid surface such as a microchip,bead. or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Suchimmobilized genotyping oligonucleotides may be used in a variety ofpolymorphism detection assays, including but not limited to probehybridization and polymerase extension assays. Immobilized TCF1genotyping oligonucleotides of the invention may comprise an orderedarray of oligonucleotides designed to rapidly screen a DNA sample forpolymorphisms in multiple genes at the same time.

An allele-specific oligonucleotide primer of the invention has a 3′terminal nucleotide, or preferably a 3′ penultimate nucleotide, that iscomplementary to only one nucleotide of a particular SNP, thereby actingas a primer for polymerase-mediated extension only if the allelecontaining that nucleotide is present. Allele-specific oligonucleotideprimers hybridizing to either the coding or noncoding strand arecontemplated by the invention. An ASO primer for detecting TCF1 genepolymorphisms could be developed using techniques known to those ofskill in the art.

Other genotyping oligonucleotides of the invention hybridize to a targetregion located one to several nucleotides downstream of one of the novelpolymorphic sites identified herein. Such oligonucleotides are useful inpolymerase-mediated primer extension methods for detecting one of thenovel polymorphisms described herein and therefore such genotypingoligonucleotides are referred to herein as “primer-extensionoligonucleotides”. In a preferred embodiment, the 3′-terminus of aprimer-extension oligonucleotide is a deoxynucleotide complementary tothe nucleotide located immediately adjacent to the polymorphic site.

In another embodiment, the invention provides a kit comprising at leasttwo genotyping oligonucleotides packaged in separate containers. The kitmay also contain other components such as hybridization buffer (wherethe oligonucleotides are to be used as a probe) packaged in a separatecontainer. Alternatively, where the oligonucleotides are to be used toamplify a target region, the kit may contain, packaged in separatecontainers, a polymerase and a reaction buffer optimized for primerextension mediated by the polymerase, such as PCR.

The above described oligonucleotide compositions and kits are useful inmethods for genotyping and/or haplotyping the TCF1 gene in anindividual. As used herein, the terms “TCF1 genotype” and “TCF1haplotype” mean the genotype or haplotype containing the nucleotide pairor nucleotide, respectively, that is present at one or more of the novelpolymorphic sites described herein and may optionally also include thenucleotide pair or nucleotide present at one or more additionalpolymorphic sites in the TCF1 gene. The additional polymorphic sites maybe currently known polymorphic sites or sites that are subsequentlydiscovered.

One embodiment of the genotyping method involves isolating from theindividual a nucleic acid mixture comprising the two copies of the TCF1gene, or a fragment thereof, that are present in the individual, anddetermining the identity of the nucleotide pair at one or more of thepolymorphic sites in the two copies to assign a TCF1 genotype to theindividual. As will be readily understood by the skilled artisan, thetwo “copies” of a gene in an individual may be the same allele or may bedifferent alleles. In a particularly preferred embodiment, thegenotyping method comprises determining the identity of the nucleotidepair at each polymorphic site.

Typically, the nucleic acid mixture is isolated from a biological sampletaken from the individual, such as a blood sample or tissue sample.Suitable tissue samples include whole blood, semen, saliva, tears,urine, fecal material, sweat, buccal smears, skin and hair. The nucleicacid mixture may be comprised of genomic DNA, mRNA, or cDNA and, in thelatter two cases, the biological sample must be obtained from an organin which the TCF1 gene is expressed. Furthermore it will be understoodby the skilled artisan that mRNA or cDNA preparations would not be usedto detect polymorphisms located in introns or in 5′ and 3′nontranscribed regions. If a TCF1 gene fragment is isolated, it mustcontain the polymorphic site(s) to be genotyped.

One embodiment of the haplotyping method comprises isolating from theindividual a nucleic acid molecule containing only one of the two copiesof the TCF1 gene, or a fragment thereof, that is present in theindividual and determining in that copy the identity of the nucleotideat one or more of the polymorphic sites in that copy to assign a TCF1haplotype to the individual. The nucleic acid may be isolated using anymethod capable of separating the two copies of the TCF1 gene orfragment, including but not limited to, one of the methods describedabove for preparing TCF1 isogenes, with targeted in vivo cloning beingthe preferred approach. As will be readily appreciated by those skilledin the art, any individual clone will only provide haplotype informationon one of the two TCF1 gene copies present in an individual. Ifhaplotype information is desired for the individual's other copy,additional TCF1 clones will need to be examined. Typically, at leastfive clones should be examined to have more than a 90% probability ofhaplotyping both copies of the TCF1 gene in an individual. In aparticularly preferred embodiment, the nucleotide at each of polymorphicsite is identified.

In a preferred embodiment, a TCF1 haplotype pair is determined for anindividual by identifying the phased sequence of nucleotides at one ormore of the polymorphic sites in each copy of the TCF1 gene that ispresent in the individual. In a particularly preferred embodiment, thehaplotyping method comprises identifying the phased sequence ofnucleotides at each polymorphic site in each copy of the TCF1 gene. Whenhaplotyping both copies of the gene, the identifying step is preferablyperformed with each copy of the gene being placed in separatecontainers. However, it is also envisioned that if the two copies arelabeled with different tags, or are otherwise separately distinguishableor identifiable, it could be possible in some cases to perform themethod in the same container. For example, if first and second copies ofthe gene are labeled with different first and second fluorescent dyes,respectively, and an allele-specific oligonucleotide labeled with yet athird different fluorescent dye is used to assay the polymorphicsite(s), then detecting a combination of the first and third dyes wouldidentify the polymorphism in the first gene copy while detecting acombination of the second and third dyes would identify the polymorphismin the second gene copy.

In both the genotyping and haplotyping methods, the identity of anucleotide (or nucleotide pair) at a polymorphic site(s) may bedetermined by amplifying a target region(s) containing the polymorphicsite(s) directly from one or both copies of the TCF1 gene, or fragmentthereof, and the sequence of the amplified region(s) determined byconventional methods. It will be readily appreciated by the skilledartisan that only one nucleotide will be detected at a polymorphic sitein individuals who are homozygous at that site, while two differentnucleotides will be detected if the individual is heterozygous for thatsite. The polymorphism may be identified directly, known aspositive-type identification, or by inference, referred to asnegative-type identification. For example, where a SNP is known to beguanine and cytosine in a reference population, a she may be positivelydetermined to be either guanine or cytosine for ail individualhomozygous at that site, or both guanine and cytosine, if the individualis heterozygous at that site. Alternatively, the site may be negativelydetermined to be not guanine (and thus cytosine/cytosine) or notcytosine (and thus guanine/guanine).

In addition, the identity of the allele(s) present at any of the novelpolymorphic sites described herein may be indirectly determined bygenotyping a polymorphic site not disclosed herein that is in linkagedisequilibrium with the polymorphic site that is of interest. Two sitesare said to be in linkage disequilibrium if the presence of a particularvariant at one she enhances the predictability of another variant at thesecond site (See, Stevens, J C 1999, Mol Diag 4:309-317). Polymorphicsites in linkage disequilibrium with the presently disclosed polymorphicsites may be located in regions of the gene or in other genomic regionsnot examined herein. Genotyping of a polymorphic site in linkagedisequilibrium with the novel polymorphic sites described herein may beperformed by, but is not limited to, any of the above-mentioned methodsfor detecting the identity of the allele at a polymorphic site.

The target region(s) may be amplified using any oligonucleotide-directedamplification method, including but not limited to polymerase chainreaction (PCR) (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR)(Barany et al., Proc Natl Acad Sci USA 88:189-193, 1991; WO 90/01069),and oligonucleotide ligation assay (OLA) (Landegren et al., Science241:1077-1080, 1988). Oligonucleotides useful as primers or probes insuch methods should specifically hybridize to a region of the nucleicacid that contains or is adjacent to the polymorphic site. Typically,the oligonucleotides are between 10 and 35 nucleotides in length andpreferably, between 15 and 30 nucleotides in length. Most preferably,the oligonucleotides are 20 to 25 nucleotides long. The exact length ofthe oligonucleotide will depend on many factors that are routinelyconsidered and practiced by the skilled artisan.

Other known nucleic acid amplification procedures may be used to amplifythe target region including transcription-based amplification systems(U.S. Pat. No. 5,130,238; EP 329,822; U.S. Pat. No. 5,169,766, WO89/06700) and isothermal methods (Walker et al., Proc Natl Acad Sci USA89:392-396, 1992).

A polymorphism in the target region may also be assayed before or afteramplification using one of several hybridization-based methods known inthe art. Typically, allele-specific oligonucleotides are utilized inperforming such methods. The allele-specific oligonucleotides may beused as differently labeled probe pairs, with one member of the pairshowing a perfect match to one variant of a target sequence and theother member showing a perfect match to a different variant. In someembodiments, more than one polymorphic site may be detected at onceusing a set of allele-specific oligonucleotides or oligonucleotidepairs. Preferably, the members of the set have melting temperatureswithin 5° C. and more preferably within 2° C., of each other whenhybridizing to each of the polymorphic sites being detected.

Hybridization of an allele-specific oligonucleotide to a targetpolynucleotide may be performed with both entities in solution or suchhybridization may be performed when either the oligonucleotide or thetarget polynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Allele-specific oligonucleotides may be synthesized directly on thesolid support or attached to the solid support subsequent to synthesis.Solid-supports suitable for use in detection methods of the inventioninclude substrates made of silicon, glass, plastic, paper and the like,which may be formed, for example, into wells (as in 96-well plates),slides, sheets, membranes, fibers, chips, dishes, and beads. The solidsupport may be treated, coated or derivatized to facilitate theimmobilization of the allele-specific oligonucleotide or target nucleicacid.

The genotype or haplotype for the TCF1 gene of an individual may also bedetermined by hybridization of a nucleic sample containing one or bothcopies of the gene to nucleic acid arrays and subarrays such asdescribed in WO 95/11995. The arrays would contain a battery ofallele-specific oligonucleotides representing each of the polymorphicsites to be included in the genotype or haplotype.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al., Proc Natl Acad Sci USA 82:7575,1985; Meyers et al., Science 230:1242, 1985) and proteins whichrecognize nucleotide mismatches, such as the E. coli mutS protein(Modrich P. Ann Rev Genet 25:229-253, 1991). Alternatively, variantalleles can be identified by single strand conformation polymorphism(SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries etal., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp.321-340, 1996) or denaturing gradient gel electrophoresis (DGGE)(Wartell et at., Nucl Acids Res 18:2699-2706, 1990; Sheffield et al.,Proc Natl Acad Sci USA 86:232-236, 1989).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature and include the “Genetic BitAnalysis” method (WO 92/15712) and the ligase/polymerase mediatedgenetic bit analysis (U.S. Pat. No. 5,679,524). Related methods aredisclosed in WO 91/02087, WO 90/09455, WO 95/17676, U.S. Pat. Nos.5,302,509 and 5,945,283. Extended primers containing a polymorphism maybe detected by mass spectrometry as described in U.S. Pat. No.5,605,798. Another primer extension method is allele-specific PCR(Ruafio et al., Nucl Acids Res 17:8392, 1989; Ruafio et al., Nucl AcidsRes 19, 6877-6882, 1991; WO 93/22456; Turki et al., I Clin Invest95:1635-1641, 1995). In addition, multiple polymorphic sites may beinvestigated by simultaneously amplifying multiple regions of thenucleic acid using sets of allele-specific primers as described inWallace et al. (WO 89/10414).

In a preferred embodiment, the haplotype frequency data for eachethnogeographic group is examined to determine whether it is consistentwith Hardy-Weinberg equilibrium. Hardy-Weinberg equilibrium (D. L. Hartlet al., Principles of Population Genomics, Sinauer Associates(Sunderland, Mass.), 3rd Ed., 1997) postulates that the frequency offinding the haplotype pair H₁/H₂ is equal to P_(H-W) (H₁/H₂)=2p(H₁) p(H₂) if H₁≠H₂ and P_(H-W) (H₁/H₂)=p (H₁) p (H₂) if H₁=H₂. Astatistically significant difference between the observed and expectedhaplotype frequencies could be due to one or more factors includingsignificant inbreeding in the population group, strong selectivepressure on the gene, sampling bias, and/or errors in the genotypingprocess. If large deviations from Hardy-Weinberg equilibrium areobserved in an ethnogeographic group, the number of individuals in thatgroup can be increased to see if the deviation is due to a samplingbias. If a larger sample size does not reduce the difference betweenobserved and expected haplotype pair frequencies, then one may wish toconsider haplotyping the individual using a direct haplotyping methodsuch as, for example, CLASPER System™ technology (U.S. Pat. No.5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin etal., Nucl Acids Res 24:4841-4843, 1996).

In one embodiment of this method for predicting a TCF1 haplotype pair,the assigning step involves performing the following analysis. First,each of the possible haplotype pairs is compared to the haplotype pairsin the reference population. Generally, only one of the haplotype pairsin the reference population matches a possible haplotype pair and thatpair is assigned to the individual. Occasionally, only one haplotyperepresented in the reference haplotype pairs is consistent with apossible haplotype pair for an individual, and in such cases theindividual is assigned a haplotype pair containing this known haplotypeand a new haplotype derived by subtracting the known haplotype from thepossible haplotype pair. In rare cases, either no haplotypes in thereference population are consistent with the possible haplotype pairs,or alternatively, multiple reference haplotype pairs are consistent withthe possible haplotype pairs. In such cases, the individual ispreferably haplotyped using a direct molecular haplotyping method suchas, for example, CLASPER System™ technology (U.S. Pat. No. 5,866,404),SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., NuclAcids Res 24:4841-4843, 1996).

The invention also provides a method for determining the frequency of aTCF1 genotype or TCF1 haplotype in a population. The method comprisesdetermining the genotype or the haplotype pair for the TCF1 gene that ispresent in each member of the population, wherein the genotype orhaplotype comprises the nucleotide pair or nucleotide detected at one ormore of the polymorphic sites in the TCF1 gene, including but notlimited to 483 A>G; and calculating the frequency any particulargenotype or haplotype is found in the population. The population may bea reference population, a family population, a same sex population, apopulation group, a trait population (e.g., a group of individualsexhibiting a trait of interest such as a medical condition or responseto a therapeutic treatment).

In another aspect of the invention, frequency data for TCF1 genotypesand/or haplotypes found in a reference population are used in a methodfor identifying an association between a trait and a TCF1 genotype or aTCF1 haplotype. The trait may be any detectable phenotype, including butnot limited to susceptibility to a disease or response to a treatmentThe method involves obtaining data on the frequency of the genotype(s)or haplotype(s) of interest in a reference population as well as in apopulation exhibiting the trait. Frequency data for one or both of thereference and trait populations may be obtained by genotyping orhaplotyping each individual in the populations using one of the methodsdescribed above. The haplotypes for the trait population may bedetermined directly or, alternatively, by the predictive genotype tohaplotype approach described above.

In another embodiment, the frequency data for the reference and/or traitpopulations is obtained by accessing previously determined frequencydata, which may be in written or electronic form. For example, thefrequency data may be present in a database that is accessible by acomputer. Once the frequency data is obtained, the frequencies of thegenotype(s) or haplotype(s) of interest in the reference and traitpopulations are compared. In a preferred embodiment, the frequencies ofall genotypes and/or haplotypes observed in the populations arecompared. If a particular genotype or haplotype for the TCF1 gene ismore frequent in the trait population than in the reference populationat a statistically significant amount, then the trait is predicted to beassociated with that TCF1 genotype or haplotype.

In a preferred embodiment statistical analysis is performed by the useof standard ANOVA tests with a Bonferoni correction and/or abootstrapping method that simulates the genotype phenotype correlationmany times and calculates a significance value. When many polymorphismsare being analyzed a correction to factor may be performed to correctfor a significant association that might be found by chance. Forstatistical methods for use in the methods of this invention, see:Statistical Methods in Biology, 3^(rd) edition, Bailey N T J, CambridgeUniv. Press (1997); introduction to Computational Biology, Waterman M S,CRC Press (2000) and Bioinformatics, Baxevanis A D and Ouellette B F Feditors (2001) John Wiley & Sons, Inc.

In a preferred embodiment of the method, the trait of interest is aclinical response exhibited by a patient to some therapeutic treatment,for example, response to a drug targeting TCF1 or response to atherapeutic treatment for a medical condition.

In another embodiment of the invention, a detectable genotype orhaplotype that is in linkage disequilibrium with the TCF1 genotype orhaplotype of interest may be used as a surrogate marker. A genotype thatis in linkage disequilibrium with a TCF1 genotype may be discovered bydetermining if a particular genotype or haplotype for the TCF1 gene ismore frequent in the population that also demonstrates the potentialsurrogate marker genotype than in the reference population at astatistically significant amount, then the marker genotype is predictedto be associated with that TCF1 genotype or haplotype and then can beused as a surrogate marker in place of the TCF1 genotype.

As used herein, “medical condition” includes but is not limited to anycondition or disease manifested as one or more physical and/orpsychological symptoms for which treatment is desirable, and includespreviously and newly identified diseases and other disorders.

As used herein, the term “clinical response” means any or all of thefollowing: a quantitative measure of the response, no response, andadverse response (i.e., side effects).

In order to deduce a correlation between clinical response to atreatment and a TCF1 genotype or haplotype, it is necessary to obtaindata on the clinical responses exhibited by a population of individualswho received the treatment, hereinafter the “clinical population”. Thisclinical data may be obtained by analyzing the results of a clinicaltrial that has already been run and/or the clinical data may be obtainedby designing and carrying out one or more new clinical trials.

As used herein, the term “clinical trial” means any research studydesigned to collect clinical data on responses to a particulartreatment, and includes but is not limited to phase I, phase II andphase III clinical trials. Standard methods are used to define thepatient population and to enroll subjects.

It is preferred that the individuals included in the clinical populationhave been graded for the existence of the medical condition of interest.This is important in cases where the symptom(s) being presented by thepatients can be caused by more than one underlying condition, and wheretreatment of the underlying conditions are not the same. An example ofthis would be where patients experience breathing difficulties that aredue to either asthma or respiratory infections. If both sets weretreated with an asthma medication, there would be a spurious group ofapparent non-responders that did not actually have asthma. These peoplewould affect the ability to detect any correlation between haplotype andtreatment outcome. This grading of potential patients could employ astandard physical exam or one or more lab tests. Alternatively, gradingof patients could use haplotyping for situations where there is a strongcorrelation between haplotype pair and disease susceptibility orseverity.

The therapeutic treatment of interest is administered to each individualin the trial population and each individual's response to the treatmentis measured using one or more predetermined criteria. It is contemplatedthat in many cases, the trial population will exhibit a range ofresponses and that the investigator will choose the number of respondergroups (e.g., low, medium, high) made up by the various responses. Inaddition, the TCF1 gene for each individual in the trial population isgenotyped and/or haplotyped, which may be done before or afteradministering the treatment.

After both the clinical and polymorphism data have been obtained,correlations between individual response and TCF1 genotype or haplotypecontent are created. Correlations may be produced in several ways. Inone method, individuals are grouped by their TCF1 genotype or haplotype(or haplotype pair) (also referred to as a polymorphism group), and thenthe averages and standard deviations of clinical responses exhibited bythe members of each polymorphism group are calculated.

These results are then analyzed to determine if any observed variationin clinical response between polymorphism groups is statisticallysignificant. Statistical analysis methods which may be used aredescribed in L. D. Fisher and G. vanBelle, “Biostatistics: A Methodologyfor the Health Sciences”, Wiley-Interscience (New York) 1993. Thisanalysis may also include a regression calculation of which polymorphicsites in the TCF1 gene give the most significant contribution to thedifferences in phenotype. One regression model useful in the inventionis described in the PCT Application entitled “Methods for Obtaining andUsing Haplotype Data”, filed Jun. 26, 2000.

A second method for finding correlations between TCF1 haplotype contentand clinical responses uses predictive models based on error-minimizingoptimization algorithms. One of many possible optimization algorithms isa genetic algorithm (R. Judson, “Genetic Algorithms and Their Uses inChemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K.B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997).Simulated annealing (Press et al., “Numerical Recipes in C: The Art ofScientific Computing”, Cambridge University Press (Cambridge) 1992, Ch.10), neural networks (E. Rich and K. Knight, “Artificial intelligence”,2nd Edition (McGraw-Hill, New York, 1991, Ch. 18), standard gradientdescent methods (Press et al., supra Ch. 10), or other global or localoptimization approaches (see discussion in Judson, supra) could also beused. Preferably, the correlation is found using a genetic algorithmapproach as described in PCT Application entitled “Methods for Obtainingand Using Haplotype Data”, filed Jun. 26, 2000.

Correlations may also be analyzed using analysis of variation (ANOVA)techniques to determine how much of the variation in the clinical datais explained by different subsets of the polymorphic sites in the TCF1gene. As described in PCT Application entitled “Methods for Obtainingand Using Haplotype Data”, filed Jun. 26, 2000, ANOVA is used to testhypotheses about whether a response variable is caused by or correlatedwith one or more traits or variables that can be measured (Fisher andvanBelle, supra, Ch. 10).

From the analyses described above, a mathematical model may be readilyconstructed by the skilled artisan that predicts clinical response as afunction of TCF1 genotype or haplotype content. Preferably, the model isvalidated in one or more follow-up clinical trials designed to test themodel.

The identification of an association between a clinical response and agenotype or haplotype (or haplotype pair) for the TCF1 gene may be thebasis for designing a diagnostic method to determine those individualswho will or will not respond to the treatment, or alternatively, willrespond at a lower level and thus may require more treatment, i.e., agreater dose of a drug. The diagnostic method may take one of severalforms: for example, a direct DNA test (i.e., genotyping or haplotypingone or more of the polymorphic sites in the TCF1 gene), a serologicaltest, or a physical exam measurement. The only requirement is that therebe a good correlation between the diagnostic test results and theunderlying TCF1 genotype or haplotype that is in turn correlated withthe clinical response. In a preferred embodiment, this diagnostic methoduses the predictive haplotyping method described above.

A computer may implement any or all analytical and mathematicaloperations involved in practicing the methods of the present invention.In addition, the computer may execute a program that generates views (orscreens) displayed on a display device and with which the user caninteract to view and analyze large amounts of information relating tothe TCF1 gene and its genomic variation, including chromosome location,gene structure, and gene family, gene expression data, polymorphismdata, genetic sequence data, and clinical data population data (e.g.,data on ethnogeographic origin, clinical responses, genotypes, andhaplotypes for one or more populations). The TCF1 polymorphism datadescribed herein may be stored as part of a relational database (e.g.,an instance of an Oracle database or a set of ASCII flat files). Thesepolymorphism data may be stored on the computer's hard drive or may, forexample, be stored on a CD-ROM or on one or more other storage devicesaccessible by the computer. For example, the data may be stored on oneor more databases in communication with the computer via a network.

In other embodiments, the invention provides methods, compositions, andkits for haplotyping and/or genotyping the TCF1 gene in an individual.The methods involve identifying the nucleotide or nucleotide pairpresent at nucleotide: 483 A>G in from GenBank accession number U72616.This nucleotide substitution changes the amino acid Asn 487 Ser in oneor both copies of the TCF1 gene from the individual. The compositionscontain oligonucleotide probes and primers designed to specificallyhybridize to one or more target regions containing, or that are adjacentto, a polymorphic site. The methods and compositions for establishingthe genotype or haplotype of an individual at the novel polymorphicsites described herein are useful for studying the effect of thepolymorphisms in the etiology of diseases affected by the expression andfunction of the TCF1 protein, studying the efficacy of drugs targetingTCF1, predicting individual susceptibility to diseases affected by theexpression and function of the TCF1 protein and predicting individualresponsiveness to drugs targeting TCF1.

In yet another embodiment, the invention provides a method foridentifying an association between a genotype or haplotype and a traitin preferred embodiments, the trait is susceptibility to a disease,severity of a disease, the staging of a disease or response to a drug.Such methods have applicability in developing diagnostic tests andtherapeutic treatments for all pharmacogenetic applications where thereis the potential for an association between a genotype and a treatmentoutcome including efficacy measurements, PK measurements and side effectmeasurements.

The present invention also provides a computer system for storing anddisplaying polymorphism data determined for the TCF1 gene. The computersystem comprises a computer processing unit; a display; and a databasecontaining the polymorphism data. The polymorphism data includes thepolymorphisms, the genotypes and the haplotypes identified for the TCF1gene in a reference population. In a preferred embodiment, the computersystem is capable of producing a display showing TCF1 haplotypesorganized according to their evolutionary relationships.

In another aspect, the invention provides SNP probes, which are usefulin classifying people according to their types of genetic variation. TheSNP probes according to the invention are oligonucleotides, which candiscriminate between alleles of a SNP nucleic acid in conventionalallelic discrimination assays.

As used herein, a “SNP nucleic acid” is a nucleic acid sequence, whichcomprises a nucleotide that is variable within an otherwise identicalnucleotide sequence between individuals or groups of individuals, thus,existing as alleles. Such SNP nucleic acids are preferably from about 15to about 500 nucleotides in length. The SNP nucleic acids may be part ofa chromosome, or they may be an exact copy of a part of a chromosome,e.g., by amplification of such a part of a chromosome through PCR orthrough cloning. The SNP nucleic adds are referred to hereafter simplyas “SNPs”. The SNP probes according to the invention areoligonucleotides that are complementary to a SNP nucleic acid.

As used herein, the term “complementary” means exactly complementarythroughout the length of the oligonucleotide in the Watson and Cricksense of the word.

In certain preferred embodiments, the oligonucleotides according to thisaspect of the invention are complementary to one allele of the SNPnucleic acid, but not to any other allele of the SNP nucleic acid.Oligonucleotides according to this embodiment of the invention candiscriminate between alleles of the SNP nucleic acid in various ways.For example, under stringent hybridization conditions, anoligonucleotide of appropriate length will hybridize to one allele ofthe SNP nucleic add, but not to any other allele of the SNP nucleicacid. The oligonucleotide may be labeled by a radiolabel or afluorescent label. Alternatively, an oligonucleotide of appropriatelength can be used as a primer for PCR, wherein the 3′ terminalnucleotide is complementary to one allele of the SNP nucleic acid, butnot to any other allele. In this embodiment, the presence or absence ofamplification by PCR determines the haplotype of the SNP nucleic acid

Thus, in one embodiment, the invention provides an isolatedpolynucleotide comprising a nucleotide sequence that is a polymorphicvariant of a reference sequence for the TCF1 gene or a fragment thereof.The reference sequence comprises UniGene Cluster Hs.73888 and thepolymorphic variant comprises at least one polymorphism, including butnot limited to nucleotide: 483 A>G. A particularly preferred polymorphicvariant is a naturally-occurring isoform (also referred to herein as an“isogene”) of the TCF1 gene.

Genomic and cDNA fragments of the invention comprise at least one novelpolymorphic site identified herein and have a length of at least 10nucleotides and may range up to the full length of the gene. Preferably,a fragment according to the present invention is between 100 and 3000nucleotides in length, and more preferably between 200 and 2000nucleotides in length, and most preferably between 500 and 1000nucleotides in length

In describing the polymorphic sites identified herein reference is madeto the sense strand of the gene for convenience. However, as recognizedby the skilled artisan, nucleic acid molecules containing the TCF1 genemay be complementary double stranded molecules and thus reference to aparticular site on the sense strand refers as well to the correspondingsite on the complementary antisense strand. Thus, reference may be madeto the same polymorphic site on either strand and an oligonucleotide maybe designed to hybridize specifically to either strand at a targetregion containing the polymorphic site. Thus, the invention alsoincludes single-stranded polynucleotides that are complementary to thesense strand of the TCF1 genomic variants described herein.

In a further aspect of the invention there is provided a kit for theidentification of a patient's polymorphism pattern at the TCF1polymorphic site at 483 A>G, said kit comprising a means for determininga genetic polymorphism pattern at the TCF1 polymorphic site at 483 A>G.

In a preferred embodiment, such kit may further comprise a DNA samplecollecting means.

In a preferred embodiment the means for determining a geneticpolymorphism pattern at the TCF1 polymorphic site at 483 A>G comprise atleast one TCF1 genotyping oligonucleotide. In particular, the means fordetermining a genetic polymorphism pattern at the TCF1 polymorphic siteat 483 A>G may comprise two TCF1 genotyping oligonucleotides. Also, themeans for determining a genetic polymorphism pattern at the TCF1polymorphic site at 483 A>G may comprise at least one TCF1 genotypingprimer compositon comprising at least one TCF1 genotypingoligonucleotide. In particular, the TCF1 genotyping primer compositonmay comprise at least two sets of allele specific primer pairs.Preferably, the two TCF1 genotyping oligonucleotides are packaged inseparate containers.

It is to be understood that the methods of the invention describedherein generally may further comprise the use of a kit according to theinvention. Generally, the methods of the invention may be performedex-vivo, and such ex-vivo methods are specifically contemplated by thepresent invention. Also, where a method of the invention may includesteps that may be practised on the human or animal body, methods thatonly comprise those steps which are not practised on the human or animalbody are specifically contemplated by the present invention.

Effect(s) of the polymorphisms identified herein on expression of TCF1may be investigated by preparing recombinant cells and/or organisms,preferably recombinant animals, containing a polymorphic variant of theTCF1 gene. As used herein, “expression” includes but is not limited toone or more of the following: transcription of the gene into precursormRNA; splicing and other processing of the precursor mRNA to producemature mRNA; mRNA stability; translation of the mature mRNA into TCF1protein (including codon usage and tRNA availability); and glycosylationand/or other modifications of the translation product, if required forproper expression and function.

To prepare a recombinant cell of the invention, the desired TCF1 isogenemay be introduced into the cell in a vector such that the isogeneremains extrachromosomal. In such a situation, the gene will beexpressed by the cell from the extrachromosomal location. In a preferredembodiment, the TCF1 isogene is introduced into a cell in such a waythat it recombines with the endogenous TCF1 gene present in the cell.Such recombination requires the occurrence of a double recombinationevent, thereby resulting in the desired TCF1 gene polymorphism. Vectorsfor the introduction of genes both for recombination and forextrachromosomal maintenance are known in the art, and any suitablevector or vector construct may be used in the invention. Methods such aselectroporation, particle bombardment, Calcium phosphateco-precipitation and viral transduction for introducing DNA into cellsare known in the art; therefore, the choice of method may lie with thecompetence and preference of the skilled practitioner.

Examples of cells into which the TCF1 isogene may be introduced include,but are not limited to, continuous culture cells, such as COS, NIH/3T3,and primary or culture cells of the relevant tissue type, i.e., theyexpress the TCF1 isogene. Such recombinant cells can be used to comparethe biological activities of the different protein variants.

Recombinant organisms, i.e., transgenic animals, expressing a variantTCF1 gene are prepared using standard procedures known in the art.Preferably, a construct comprising the variant gene is introduced into anonhuman animal or an ancestor of the animal at an embryonic stage,i.e., the one-cell stage, or generally not later than about theeight-cell stage. Transgenic animals carrying the constructs of theinvention can be made by several methods known to those having skill inthe art. One method involves transfecting into the embryo a retrovirusconstructed to contain one or more insulator elements, a gene or genesof interest, and other components known to those skilled in the art toprovide a complete shuttle vector harboring the insulated gene(s) as atransgene, see e.g., U.S. Pat. No. 5,610,053. Another method involvesdirectly injecting a transgene into the embryo. A third method involvesthe use of embryonic stem cells.

Examples of animals, into which the TCF1 isogenes may be introducedinclude, but are not limited to, mice, rats, other rodents, and nonhumanprimates (see “The introduction of Foreign Genes into Mice” and thecited references therein, in: Recombinant DNA, Eds. J. D. Watson, M.Gilman, J. Witkowski, and M. Zoller; W. H. Freeman and Company, NewYork, pages 254-272). Transgenic animals stably expressing a human TCF1isogene and producing human TCF1 protein can be used as biologicalmodels for studying diseases related to abnormal TCF1 expression and/oractivity, and for screening and assaying various candidate drugs,compounds, and treatment regimens to reduce the symptoms or effects ofthese diseases.

In addition, treatment with a glycemic control agent or therapy can beused in subjects with impaired glycemic control, including: type 2 andtype 1 diabetes, impaired glucose metabolism (impaired glucose toleranceand/or impaired fasting glucose), Syndrome X, prandial lipemia,gestational diabetes, for the prevention or delay of progression toovert diabetes mellitus type 2; for the prevention, reduction or delayin onset of a condition selected from the group consisting of increasedmicrovascular complications; increased cardiovascular morbidity; excesscerebrovascular diseases; increased cardiovascular mortality and suddendeath; higher incidences and mortality rates of malignant neoplasms; andother metabolic disturbances that are associated with IGM.

Furthermore, glycemic control agents or therapies can be used insubjects with impaired glycemic control (IGC) for the prevention,reduction or delay in onset of a condition selected from the group e.g.consisting of retinopathy, other ophthalmic complications of diabetes,nephropathy, neuropathy, peripheral angiopathy, peripheral angiopathy,gangrene, myocardial infarctions, coronary heart disease,atherosclerosis, other acute and subacute forms of coronary ischemia,stroke, dyslipidemia, hyperuricemia, hypertension, angina pectoris,microangiopathic changes that result in amputation, cancer, cancerdeaths, obesity, uricemia, insulin resistance, arterial occlusivedisease, and atherosclerosis.

According to the present invention, glycemic control agents or therapiesagents can be used in subjects with IGC, to prevent or delay theprogression to overt diabetes, to reduce microvascular complications ofdiabetes, to reduce vascular, especially cardiovascular, mortality andmorbidity, especially cardiovascular morbidity and mortality, and toreduce increased mortality related to cancer in individuals with IGC.

Accordingly, the present invention relates to a method in subjects withIGC, for the prevention or delay of progression to overt diabetesmellitus type 2; for the prevention, reduction or delay in onset of acondition selected from the group consisting of increased microvascularcomplications; increased cardiovascular morbidity; excesscerebrovascular diseases; increased cardiovascular mortality and suddendeath; higher incidences and mortality rates of malignant neoplasms; andother metabolic disturbances that are associated with IGC. Especially,the present invention relates to a method used in subjects with IGC, forthe prevention, reduction or delay in onset of a condition selected fromthe group e.g. consisting of retinopathy, other ophthalmic complicationsof diabetes, nephropathy, neuropathy, peripheral angiopathy, peripheralangiopathy gangrene, myocardial infarctions, coronary heart disease,atherosclerosis, other acute and subacute forms of coronary ischemia,stroke, dyslipidemia, hyperuricemia, hypertension, angina pectoris,microangiopathic changes that result in amputation, cancer, cancerdeaths, obesity, uricemia, insulin resistance, arterial occlusivedisease, and atherosclerosis.

Accordingly, the present invention relates to a method of prevention ordelay of the progression to overt diabetes, especially type 2 (ICD-9Code 250.2), prevention or reduction of microvascular complications likeretinopathy (ICD-9 code 250.5), neurophathy (ICD-9 code 250.6),nephropathy (ICD-9 code 250.4) and peripheral angiopathy or gangrene(ICD-9 code 250.7), later termed “microvascular complications” insubjects with IGM, especially IFG and IGT. Further the present inventionrelates to a method to prevent or reduce conditions of excessivecardiovascular morbidity (ICD-9 codes 410-414), e.g. myocardialinfarction (ICD-9 code 410), arterial occlusive disease, atherosclerosisand other acute and subacute forms of coronary ischemia (ICD-9 code411-414), later termed “cardiovascular morbidity”; to prevent, reduce,or delay the onset of excess cerebrovascular diseases like stroke (ICD-9codes 430-438); to reduce increased cardiovascular mortality (ICD-9codes 390-459) and sudden death (ICD-9 code 798.1); to prevent thedevelopment of cancer (ICD-9 codes 140-208) and to reduce cancer deaths,in each case, in subjects with IGC.

The method further relates to a method of prevention or reduction ofother metabolic disturbances that are associated with IGC includinghyperglycemia (including isolated postprandial hyperglycemia),dyslipidemia (ICD-9 code 272), hyperuricemia (ICD-9 code 790.6) as wellas hypertension (ICD-9 codes 401-404) and angina pectoris (ICD-9 code413.9), in each case, in subjects with IGC. The codes identifiedhereinbefore and herafter according to the international Classificationof Diseases 9th version and the corresponding definitions allocatedthereto are herewith incorporated by reference and likewise form part ofthe present invention.

The method comprises administering to a subject in need thereof aneffective amount of a glycemic control agents or therapies or apharmaceutically acceptable salt of such an agent or compound. A subjectin need of such method is a warm-blooded animal including man. Thepresent invention also relates to a method to be used in subjects withIGC, and associated diseases and conditions such as isolated prandialhyperglycemia, prevention or delay of the progression to overt diabetes,especially type 2, prevention, reduction, or delay the onset ofmicrovascular complications, prevention or reduction of gangrene ormicroangiopathic changes that result in amputation, prevention orreduction of excessive cardiovascular morbidity and cardiovascularmortality, prevention of cancer and reduction of cancer deaths.

The present invention likewise relates to a method of treatment ofconditions and diseases associated with IGC (including isolated prandialhyperglycemia) including obesity, increased age, diabetes duringpregnancy, dyslipidemia, high blood pressure, uricemia, insulinresistance, arterial occlusive disease, atherosclerosis, retinopathy,nephropathy, angina pectoris, myocardial infarction, and stroke.Preferably, said preventions should be effected in individuals withglucose levels in the ranges that have been proven in largeepidemiologic studies to confer increased cardiovascular, microvascularand cancer risk. These levels include levels of plasma glucose 7.8mmol/L mmol/L after an OGTT or casual glucose evaluation and/or fastingplasma glucose in the IFG range (fasting plasma glucose between 6.1 and7 mmol/L). As new epidemiologic data become available to lower theglycemic levels that are incontrovertibly linked to the above-mentionedrisks, or as the international standards for defining the risk groupsare changed, the use of the invention is also warranted for treatment ofthe groups at risk.

The present invention also relates to a method to be used in subjectswith IFG comprising administering to a subject in need thereof atherapeutically effective amount of a glycemic control agents, includingbut not limited to a DPP-IV inhibitor.

The present invention relates to the use of a glycemic control agents ora pharmaceutically acceptable salt thereof for the manufacture of amedicament in subjects with IGC, for the prevention or delay ofprogression to overt diabetes mellitus type 2; for the prevention,reduction or delay in onset of a condition selected from the groupconsisting of increased microvascular complications; increasedcardiovascular morbidity; excess cerebrovascular diseases; increasedcardiovascular mortality and sudden death; higher incidences andmortality rates of malignant neoplasms; and other metabolic disturbancesthat are associated with IGC.

The present invention relates to the use of an glycemic control agentincluding a DPP4 inhibitor or a pharmaceutically acceptable salt for themanufacture of a medicament in subjects with IGC, and associateddiseases and conditions such as isolated prandial hyperglycemia for thefollowing: prevention or delay of the progression to overt diabetes,especially type 2, prevention or reduction of microvascularcomplications, prevention or reduction of excessive cardiovascularmorbidity and cardiovascular mortality, prevention of cancer andreduction of cancer deaths.

The corresponding active ingredient or a pharmaceutically acceptablesalt thereof may also be used in form of a hydrate or include othersolvents used for crystallization. Furthermore, the present inventionrelates to the combination such as a combined preparation orpharmaceutical composition, respectively, comprising more than oneglycemic control agents to be used in subjects with IGM, especially IFGand/or IGT, for the prevention or delay of progression to overt diabetesmellitus type 2; for the prevention, reduction or delay in onset of acondition selected from the group consisting of increased microvascularcomplications; increased cardiovascular morbidity; excesscerebrovascular diseases; increased cardiovascular mortality and suddendeath; higher incidences and mortality rates of malignant neoplasms; andother metabolic disturbances that are associated with IGM.

Further benefits when applying the combination of the present inventionare that lower doses of the individual drugs to be combined according tothe present invention can be used to reduce the dosage, for example,that the dosages need not only often be smaller but are also appliedless frequently, or can be used in order to diminish the incidence ofside effects. This is in accordance with the desires and requirements ofthe patients to be treated. Preferably, the jointly therapeuticallyeffective amounts of the active agents according to the combination ofthe present invention can be administered simultaneously or sequentiallyin any order, separately or in a fixed combination.

The term “therapeutically effective amount” as used herein, shall meanthat amount of a drug or combination that will elicit the biological ormedical response needed to achieve the therapeutic effect as specifiedaccording to the present invention in the warm-blooded animal, includingman. A “therapeutically effective amount” can be administered whenadministering a single agent and also in both a fixed or freecombination of two or more compounds.

A “jointly effective amount” as used herein, shall mean an amount of oneor more components of a combination that may be non-effective by itselfbut when used in a combination according to the present invention may betherapeutically effective in combination with one or more other agentsif the overall therapeutic effect can be achieved by the combinedadministration of the (fixed or free) multiple agents. Thepharmaceutical composition according to the present invention asdescribed hereinbefore and hereinafter may be used for simultaneous useor sequential use in any order, for separate use or as a fixedcombination.

Preferred glycemic control agents include, but are not limited to, DPP4inhibitors such as the compounds; 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S) and(1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile)or, if appropriate, in each case, a pharmaceutically acceptable saltthereof.

In a variation thereof, the present invention likewise relates to a“kit-of-parts”, for example, in the sense that the components to becombined according to the present invention can be dosed independentlyor by use of different fixed combinations with distinguished amounts ofthe components, i.e. simultaneously or at different time points. Theparts of the kit of parts can then e.g. be administered simultaneouslyor chronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts.Preferably, the time intervals are chosen such that the effect on thetreated disease or condition in the combined use of the parts is largerthan the effect that would be obtained by use of only any one of thecomponents. The invention furthermore relates to a commercial packagecomprising the combination according to the present invention togetherwith instructions for simultaneous, separate or sequential use. Thecompounds to be combined can be present as pharmaceutically acceptablesalts. If these compounds have, for example, at least one basic center,they can form acid addition salts. Corresponding acid addition salts canalso be formed having, if desired, an additionally present basic center.The compounds having an acid group (for example COOH) can also formsalts with bases. Pharmaceutically acceptable salts are for example,salts formed with bases, namely cationic salts such as alkali andalkaline earth metal salts, as well as ammonium salts.

The pharmaceutical compositions according to the invention can beprepared in a manner known per se and are those suitable for enteral,such as oral or rectal, and parenteral administration to mammals(warm-blooded animals), including man, comprising a therapeuticallyeffective amount of the pharmacologically active compound, alone or incombination with one or more pharmaceutically acceptable carries,especially suitable for enteral or parenteral application.

The novel pharmaceutical preparations contain, for example, from about10% to about 100%, preferably 80%, most preferably from about 90% toabout 99%, of the active ingredient. Pharmaceutical preparationsaccording to the invention for enteral or parenteral administration are,for example, those in unit dose forms, such as sugar-coated tablets,tablets, capsules or suppositories, or ampoules. These are prepared in amanner well known to one of skill in the art, for example by means ofconventional mixing, granulating, sugarcoating, dissolving orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active ingredient with solid carriers,if desired granulating a mixture obtained, and processing the mixture orgranules, if desired or necessary, after addition of suitable excipientsto give tablets or sugar-coated tablet cores.

The precise dosage of the compounds of the present invention, and theircorresponding pharmaceutically acceptable acid addition salts, to beemployed for treating conditions or disorders characterized by impairedglycemic control depends upon several factors, including the host, thenature and the severity of the condition being treated, the mode ofadministration and the particular compound employed. However, ingeneral, conditions or disorders characterized by impaired glycemiccontrol are effectively treated when a compound of the invention, or acorresponding pharmaceutically acceptable acid addition salt, isadministered enterally, e.g., orally, or parenterally, e.g.,intravenously, but preferably orally, at a daily dosage of 0.002-10mg/kg body weight, preferably 0.02-2.5 mg/kg body weight or, for mostlarger primates, a daily dosage of 0.1-250, preferably 1-100 mg. Atypical oral dosage unit is 0.01-0.75 mg/kg, one to three times a day.

Usually, a small dose is administered initially and the dosage isgradually increased until the optimal dosage for the host undertreatment is determined. The upper limit of dosage is that imposed byside effects and can be determined by trial for the host being treated.

The compounds of the present invention, and their correspondingpharmaceutically acceptable acid addition salts, may be combined withone or more pharmaceutically acceptable carriers and, optionally, one ormore other conventional pharmaceutical adjuvants and administeredenterally, e.g., orally, in the form of tablets, capsules, caplets, etc.or parenterally, e.g., intravenously, in the form of sterile injectablesolutions or suspensions. The enteral and parenteral compositions may beprepared by conventional means.

The compounds of the present invention, and their correspondingpharmaceutically acceptable acid addition salts, may be formulated intoenteral and parenteral pharmaceutical compositions containing an amountof the active substance that is effective for treating conditions ordisorders characterized by impaired glycemic control and apharmaceutically acceptable carrier such compositions may be formulatedin unit dosage form.

The compounds of the present invention (including those of each of thesubscopes thereof and each of the examples) may be administered inenantiomerically pure form (e.g., purity greater that 98% and preferablygreater than 99% of one enantiomer) or with both enantiomers presenttogether, e.g., in racemic form. The above dosage ranges are based on asingle enantiomer of the compounds of the present invention. (excludingthe amount of the less active enantiomer, if any).

A person skilled in the art is fully enabled, based on his knowledge, todetermine the specific doses for the specific glycemic control agent,including DPP4 inhibitors, whether taken alone or in combination.

EXAMPLES

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

Example 1

A 40 year old woman is found, on routine screening, to have an elevatedblood glucose level. Her physician performs an oral glucose tolerancetest and determines that the patient has impaired glucose tolerance. Thephysician discusses with the patient the short- and long-termconsequences of impaired glucose tolerance and the possibility ofprogression to overt diabetes. The physician also discusses theavailable treatment modalities including diet, weight loss, exercise andmedications including various glycemic control agents such as the DPP4inhibitors then available. In addition, the physician counsels thepatient about the possibility of testing her for the presence of thepolymorphism in the TCF1 gene and explains what this result would meanwith regard to the use of medication, including DPP4 inhibitors.

The patient agrees to the testing and the genotyping shows the presenceof the GG genotype. On the basis of these results, the physicianrecommends and the patient agrees to a trial of a medication such as aDPP4 inhibitor to help correct her abnormal glucose tolerance andpost-prandial hyperglycemia.

Example 2

A 52 year old man with type II diabetes is seen by his physician. Thepatient is taking a glycemic control agent and glucose levels are ingood control but the patient is experiencing numerous side effects fromthe medication. The physician recommends genotyping and counsels thepatient regarding the treatment options that the genotyping resultswould allow. The patient is tested and determined to have the genotypeassociated with the most favorable response to DPP4 inhibitors. On thebasis of this result and the expected high sensitivity to DDP4inhibitors the physician is able to recommend a treatment regimen with alow dose of a DPP4 inhibitor with reduced likelihood of side effects.This treatment can supplement continued treatment with a reduced dose ofthe glycemic control agent this patient was previously treated with andwas not able to tolerate or a low dose regimen of the DPP4 inhibitoralone can be substituted.

Definitions

As used herein, in the context of this disclosure, the following termsshall be defined as follows unless otherwise indicated:

Allele—A particular form of a genetic locus, distinguished from otherforms by its particular nucleotide sequence.

Candidate gene—A gene which is hypothesized to be responsible for adisease, condition, or the response to a treatment, or to be correlatedwith one of these.

Gene—A segment of DNA that contains all the information for theregulated biosynthesis of an RNA product, including promoters, exons,introns, and other untranslated regions that control expression.

Genotype—An unphased 5′ to 3′ sequence of nucleotide pair(s) found atone or more polymorphic sites in a locus on a pair of homologouschromosomes in an individual. As used herein, genotype includes afull-genotype and/or a sub-genotype as described below.

Full-genotype—The unphased 5′ to 3′ sequence of nucleotide pairs foundat all known polymorphic sites in a locus on a pair of homologouschromosomes in a single individual.

Sub-genotype—The unphased 5′ to 3′ sequence of nucleotides seen at asubset of the known polymorphic sites in a locus on a pair of homologouschromosomes in a single individual.

Genotyping—A process for determining a genotype of an individual.

Haplotype—A 5′ to 3′ sequence of nucleotides found at one or morepolymorphic sites in a locus on a single chromosome from a singleindividual. As used herein, haplotype includes a full-haplotype and/or asub-haplotype as described below.

Full-haplotype—The 5′ to 3′ sequence of nucleotides found at all knownpolymorphic sites in a locus on a single chromosome from a singleindividual.

Sub-haplotype—The 5′ to 3′ sequence of nucleotides seen at a subset ofthe known polymorphic sites in a locus on a single chromosome from asingle individual.

Haplotype pair—The two haplotypes found for a locus in a singleindividual.

Haplotyping—A process for determining one or more haplotypes in anindividual and includes use of family pedigrees, molecular techniquesand/or statistical inference.

Haplotype data—information concerning one or more of the following for aspecific gene: a listing of the haplotype pairs in each individual in apopulation; a listing of the different haplotypes in a population;frequency of each haplotype in that or other populations, and any knownassociations between one or more haplotypes and a trait.

Isoform—A particular form of a gene, mRNA, cDNA or the protein encodedthereby, distinguished from other forms by its particular sequenceand/or structure.

Isogene—One of the isoforms of a gene found in a population. An isogenecontains all of the polymorphisms present in the particular isoform ofthe gene.

Isolated—As applied to a biological molecule such as RNA, DNA,oligonucleotide, or protein, isolated means the molecule issubstantially free of other biological molecules such as nucleic acids,proteins, lipids, carbohydrates, or other material such as cellulardebris and growth media. Generally, the term “isolated” is not intendedto refer to a complete absence of such material or to absence of water,buffers, or salts, unless they are present in amounts that substantiallyinterfere with the methods of the present invention.

Linkage—describes the tendency of genes to be inherited together as aresult of their location on the same chromosome; measured by percentrecombination between loci.

Linkage disequilibrium—describes a situation in which some combinationsof genetic markers occur more or less frequently in the population thanwould be expected from their distance apart. It implies that a group ofmarkers has been inherited coordinately. It can result from reducedrecombination in the region or from a founder effect, in which there hasbeen insufficient time to reach equilibrium since one of the markers wasintroduced into the population.

Locus—A location on a chromosome or DNA molecule corresponding to a geneor a physical or phenotypic feature.

Naturally-occurring—A term used to designate that the object it isapplied to, e.g., naturally-occurring polynucleotide or polypeptide, canbe isolated from a source in nature and which has not been intentionallymodified by man.

Nucleotide pair—The nucleotides found at a polymorphic site on the twocopies of a chromosome from an individual.

Phased—As applied to a sequence of nucleotide pairs for two or morepolymorphic sites in a locus, phased means the combination ofnucleotides present at those polymorphic sites on a single copy of thelocus is known.

Polymorphic site (PS)—A position within a locus at which at least twoalternative sequences are found in a population, the most frequent ofwhich has a frequency of no more than 99%.

Polymorphic variant—A gene, mRNA, cDNA, polypeptide or peptide whosenucleotide or amino acid sequence varies from a reference sequence dueto the presence of a polymorphism in the gene.

Polymorphism—The sequence variation observed in an individual at apolymorphic site. Polymorphisms include nucleotide substitutions,insertions, deletions and microsatellites and may, but need not, resultin detectable differences in gene expression or protein function.

Polymorphism data—information concerning one or more of the followingfor a specific gene: location of polymorphic sites; sequence variationat those sites; frequency of polymorphisms in one or more populations;the different genotypes and/or haplotypes determined for the gene;frequency of one or more of these genotypes and/or haplotypes in one ormore populations; any known association(s) between a trait and agenotype or a haplotype for the gene.

Polymorphism database—A collection of polymorphism data arranged in asystematic or methodical way and capable of being individually accessedby electronic or other means.

Polynucleotide—A nucleic acid molecule comprised of single-stranded RNAor DNA or comprised of complementary, double-stranded DNA.

Population group—A group of individuals sharing a common characteristicsuch as ethnogeographic origin, medical condition, response to treatmentetc.

Reference population—A group of subjects or individuals who arepredicted to be representative of 1 or more characteristics of thepopulation group. Typically, the reference population represents thegenetic variation in the population at a certainty level of at least85%, preferably at least 90%, more preferably at least 95% and even morepreferably at least 99%.

Single Nucleotide Polymorphism (SNP)—Typically, the specific pair ofnucleotides observed at a single polymorphic site. In rare cases, threeor four nucleotides may be found.

Subject—A human individual whose genotypes or haplotypes or response totreatment or disease state are to be determined.

Treatment—A stimulus administered internally or externally to a subject.

Unphased—As applied to a sequence of nucleotide pairs for two or morepolymorphic sites in a locus, unphased means the combination ofnucleotides present at those polymorphic sites on a single copy of thelocus is not known.

DPP4 inhibitor—as used herein, the term DPP4 inhibitor means a compoundcapable of inhibiting the catalytic actions of the enzyme DPP4 (DPP-IV;dipeptidylpeptidase IV; EC 3.4.14.5), which is a serine exopeptidaseidentical to ADA complexing protein-2 and to the T-cell activationantigen CD26.

References Cited

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. The discussion of references herein isintended merely to summarise the assertions made by their authors and noadmission is made that any reference constitutes prior art. Applicantsreserve the right to challenge the accuracy and pertinence of the citedreferences.

In addition, all GenBank accession numbers, Unigene Cluster numbers andprotein accession numbers cited herein are incorporated herein byreference in their entirety and for all purposes to the same extent asif each such number was specifically and individually indicated to beincorporated by reference in its entirety for all purposes

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatus within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications and variations are intended to fall withinthe scope of the appended claims. The present invention is to be limitedonly by the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A method for determining the responsiveness of an individual with adisorder characterized by impaired glycemic control to treatment with aglycemic control agent or therapy, comprising; (a) determining for thetwo copies of the TCF1 gene present in the individual, the identity ofthe nucleotide pair at the polymorphic site at 483 A>G, and (b)assigning the individual to a good responder group if both pairs are GCor if one pair is AT and one pair is GC and to a low responder group ifboth pairs are AT.
 2. The method of claim 1 wherein the glycemic controlagent or therapy comprises administration of a dipeptidylpeptidase 4(DPP4) inhibitor.
 3. The method of claim 1 wherein the glycemic controlagent or therapy comprises administration of 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S).
 4. Themethod of claim 1 wherein the glycemic control agent or therapycomprises administration of1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile. 5.The method of claim 1 wherein the glycemic control agent or therapy isselected from the compounds of Formula I or Formula II.
 6. The method ofclaim 1 wherein the disorder characterized by impaired glycemic controlis type 2 diabetes mellitus.
 7. The method of claim 1 wherein thedisorder characterized by impaired glycemic control is type 1 diabetesmellitus.
 8. The method of claim 1 wherein the disorder characterized byimpaired glycemic control is impaired glucose tolerance.
 9. The methodof claim 1 wherein the disorder characterized by impaired glycemiccontrol is impaired fasting glucose.
 10. The method of claim 1 whereinthe disorder characterized by impaired glycemic control is Syndrome X.11. The method of claim 1 wherein the disorder characterized by impairedglycemic control is gestational diabetes.
 12. The method of claim 1wherein the disorder characterized by impaired glycemic control isimpaired glucose metabolism (IGM).
 13. The method of claim 1 wherein thedisorder characterized by impaired glycemic control is a disorderresponsive to DPP4 inhibitors
 14. A method for treating an individualwith a disorder characterized by impaired glycemic control, comprising,(a) determining, for the two copies of the TCF1 gene present in theindividual, the identity of the nucleotide pair at the polymorphic site483 A>G, wherein, (b) if both the nucleotide pairs are CG or if one isAT and one is CG the individual is treated with a glycemic control agentor therapy and if the nucleotide pairs are both AT the individual istreated with alternate therapy.
 15. The method of claim 14 wherein theglycemic control agent or therapy comprises administration of adipeptidylpeptidase 4 (DPP4) inhibitor.
 16. The method of claim 14wherein the glycemic control agent or therapy comprises administrationof 2-Pyrrolidinecarbonitrile, 1-[[[2-[(5-cyano-2-pyridinyl)amino]ethyl]amino]acetyl]-, (2S).
 17. The method of claim 14 wherein theglycemic control agent or therapy comprises administration of1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile.18. The method of claim 14 wherein the glycemic control agent isselected from the compounds of Formula I or Formula II.
 19. The methodof claim 14 wherein the disorder characterized by impaired glycemiccontrol is type 2 diabetes mellitus.
 20. The method of claim 14 whereinthe disorder characterized by impaired glycemic control is type 1diabetes mellitus.
 21. The method of claim 14 wherein the disordercharacterized by impaired glycemic control is impaired glucosetolerance.
 22. The method of claim 14 wherein the disorder characterizedby impaired glycemic control is impaired fasting glucose.
 23. The methodof claim 14 wherein the disorder characterized by impaired glycemiccontrol is Syndrome X.
 24. The method of claim 14 wherein the disordercharacterized by impaired glycemic control is gestational diabetes. 25.The method of claim 14 wherein the disorder characterized by impairedglycemic control is impaired glucose metabolism (IGM).
 26. The method ofclaim 14 wherein the disorder characterized by impaired glycemic controlis a disorder responsive to DPP4 inhibitors.
 27. A method foridentifying an association between a trait and at least one genotype orhaplotype of the TCF1 gene which comprises, comparing the frequency ofthe genotype or haplotype in a population exhibiting the trait with thefrequency of the genotype or haplotype in a reference population,wherein the genotype or haplotype comprises a nucleotide pair ornucleotide located at the polymorphic site 483 A>G, wherein a higherfrequency of the genotype or haplotype in the trait population than inthe reference population indicates the trait is associated with thegenotype or haplotype.
 28. The method of claim 26, wherein the trait isa clinical response to a drug targeting TCF1 or DPP4.
 29. A method fortreating an individual, with a disorder characterized by impairedglycemic control, comprising, (a) determining, for the two copies of theTCF1 gene present in the individual, the identity of the nucleotide pairat the polymorphic site 483 A>G, wherein, (b) if both the nucleotidepairs are CG or if one is AT and one is CG the individual is treatedwith a low dose of a glycemic control agent and if the nucleotide pairsare both AT the individual is treated with a high dose of a glycemiccontrol agent.
 30. The method of claim 29 wherein the glycemic controlagent is a dipeptidylpeptidase 4 (DPP4) inhibitor.
 31. The method ofclaim 29 wherein the glycemic control agent or therapy comprisesadministration of 2-Pyrrolidinecarbonitrile,1-[[[2-[(5-cyano-2-pyridinyl) amino]ethyl]amino]acetyl]-, (2S).
 32. Themethod of claim 29 wherein the glycemic control agent or therapycomprises administration of1-[3Hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile.33. The method of claim 29 wherein the glycemic control agent or therapyis selected from the compounds of Formula I or Formula II.
 34. Themethod of claim 29 wherein the disorder characterized by impairedglycemic control is type 2 diabetes mellitus.
 35. The method of claim 29wherein the disorder characterized by impaired glycemic control is type1 diabetes mellitus.
 36. The method of claim 29 wherein the disordercharacterized by impaired glycemic control is impaired glucosetolerance.
 37. The method of claim 29 wherein the disorder characterizedby impaired glycemic control is impaired fasting glucose.
 38. The methodof claim 29 wherein the disorder characterized by impaired glycemiccontrol is Syndrome X.
 39. The method of claim 29 wherein the disordercharacterized by impaired glycemic control is gestational diabetes. 40.The method of claim 29 wherein the disorder characterized by impairedglycemic control is impaired glucose metabolism (IGM).
 41. The method ofclaim 29 wherein the disorder characterized by impaired glycemic controlis a disorder responsive to DPP4 inhibitors.
 42. A method of treating apatient with a disorder characterized by impaired glycemic controlcomprising, (a) providing genetic counseling to the patient and patientsfamily, (b) determining the patients genotype for the TCF1 gene at thepolymorphism site 483 A>G, (c) determining the proper therapy for saidpatient based on results of the genotype determination.
 43. A method foroptimizing clinical trial design for glycemic control agents,comprising, (a) determining, for the two copies of the TCF1 gene presentin an individual being considered for inclusion in the clinical trial,the identity of the nucleotide pair at the polymorphic site 483 A>Gwherein, (b) if both the nucleotide pairs are CG or if one is AT and oneis CG the individual is included in the clinical trial and if thenucleotide pairs are both AT the individual is not included.
 44. Amethod for identifying individuals, with a disorder characterized byimpaired glycemic control, who would benefit from drug A vs. B,comprising, (a) determining, for the two copies of the TCF1 gene presentin the individual, the identity of the nucleotide pair at thepolymorphic site 483 A>G, wherein, (b) both the nucleotide pairs are CGor if one is AT and one is CG the individual would benefit from aglycemic control agent or therapy and if the nucleotide pairs are bothAT the individual would benefit from alternate glycemic control therapy.45. A method for determining which individuals, with a disordercharacterized by impaired glycemic control, could be treated with aglycemic control agents with reduced side effects, comprising,determining, for the two copies of the TCF1 gene present in theindividual, the identity of the nucleotide pair at the polymorphic site483 A>G, wherein, if both the nucleotide pairs are CG or if one is ATand one is CG the individual can be treated with lower doses of aglycemic control agent with fewer side effects and if the nucleotidepairs are both AT the individual must be treated with higher doses of aglycemic control agent and therefore greater side effects.
 46. A methodfor determining the responsiveness of an individual with a disordercharacterized by impaired glycemic control to treatment with a glycemiccontrol agent or therapy, comprising; (a) determining, for the twocopies of the TCF1 gene present in the individual, the identity of anucleotide pair at a polymorphic site in the region of the TCF1 genethat is in linkage disequilibrium with the polymorphic site at TCF1 483A>G, and (b) assigning the individual to a good responder group if thenucleotide pair at a polymorphic site in the region of the TCF1 genethat is in linkage disequilibrium with the polymorphic site at 483 A>Gindicates that, at the TCF1 polymorphic site at 483 A>G, both nucleotidepairs are GO or one pair is AT and one pair is GC and to a low respondergroup if said nucleotide pair indicates that both pairs are AT at theTCF1 483 A>G site.
 47. A kit for the identification of a patient'spolymorphism pattern at the TCF1 polymorphic site at 483 A>G, said kitcomprising a means for determining a genetic polymorphism pattern at theTCF1 polymorphic site at 483 A>G.
 48. A kit according to claim 47,further comprising a DNA sample collecting means.
 49. A kit according toclaim 47, wherein the means for determining a genetic polymorphismpattern at the TCF1 polymorphic site at 483 A>G comprise at least oneTCF1 genotyping oligonucleotide.
 50. A kit according to claim 47 whereinthe means for determining a genetic polymorphism pattern at the TCF1polymorphic site at 483 A>G comprise two TCF1 genotypingoligonucleotides.
 51. A kit according to claim 47, wherein the means fordetermining a genetic polymorphism pattern at the TCF1 polymorphic siteat 483 A>G comprise at least one TCF1 genotyping primer compositorcomprising at least one TCF1 genotyping oligonucleotide.
 52. A kitaccording to claim 51, wherein the TCF1 genotyping primer compositorcomprises at least two sets of allele specific primer pairs.
 53. A kitaccording to claim 50, wherein the two TCF1 genotyping oligonucleotidesare packaged in separate containers.
 54. A method according to claim 1,wherein the determination step (a) further comprises the use a kitaccording to claim
 47. 55. A method according to claim 1, wherein saidmethod is performed ex vivo.